Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Molecular Profiling for Bethesda III to VI Nodules: Results of a Multicenter International Retrospective Study.

OBJECTIVE: Molecular testing is a well-established tool that assists in the management of thyroid nodules. We describe our experience using molecular testing of thyroid nodules with Bethesda III to VI cytology. METHODS: This is a retrospective multicenter, multinational study of thyroid nodules that underwent preoperative molecular profiling with ThyGenX/ThyGeNEXT or ThyroSeq V3 between 2015 and 2022. The clinical characteristics and mutational profiles of tumors were compared. Collected data included demographics, cytology results, surgical pathology, and molecular alterations. Molecular alterations were categorized into 3 main phenotypes: BRAF-like, RAS-like, and non-BRAF-non-RAS (NBNR). RESULTS: Overall, 784 patients who had surgery were included, of which 603 (76.2%) were females. The most common histologic type was papillary thyroid cancer (PTC) with 727 (91.9%) cases. In total, 205 (28.2%) cases showed an aggressive subtype of PTC (eg, tall cell and hobnail). BRAF-like alterations were most likely to be found in Bethesda V and VI nodules and show extrathyroidal extension (ETE), nodal disease, and/or aggressive subtypes of PTC (P < .001 for all). RAS-like alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive histology (P < .001 for all). NBNR alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive subtypes of PTC. However, they were rarely but significantly associated with poorly differentiated thyroid cancer (P < .005). CONCLUSION: Molecular testing of thyroid nodules can help determine the likelihood of malignancy and classify nodules into several tumor phenotypes, predicting their behaviors and potentially allowing for a more tailored treatment. NBNR alterations should be managed with caution.

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

Outcome and racing performance following standing fracture repair in 245 horses.

BACKGROUND: Repair of sagittal proximal phalanx (P1) and parasagittal metacarpal/metatarsal III (MC/MTIII) fractures has evolved over recent decades from a procedure carried out solely under general anaesthesia, to one commonly performed under standing sedation. To date, standing fracture repair has not been evaluated for large cohorts. OBJECTIVES: To determine short-term (survival to discharge) and long-term (return to racing) outcomes of horses undergoing standing repair of MC/MTIII and P1 fractures, and to compare pre-surgical and post-surgical racing performance. STUDY DESIGN: Single-centre retrospective cohort study. METHODS: Retrospective clinical record review of 245 cases undergoing standing repair of MC/MTIII or P1 fractures, 1 January 2007-30 June 2021. Data on signalment, fracture configuration and complications were collected and full race records were retrieved from the Racing Post Database (wwww.racingpost.com). Chi-squared and Mann-Whitney U tests were used to determine any difference in variables between horses that raced after surgery compared to those that did not. McNemar change and Wilcoxon signed-rank tests were used to compare pre- and post-surgical racing performance, p ≤ 0.05. RESULTS: Ninety-eight percent [95% confidence interval (CI): 96.2%-99.7%] of horses survived hospital discharge, and 75.1% (95% CI: 68.9%-81.4%) raced after surgery, a median of 241 days later. Horses that raced post-surgery were significantly less likely to have suffered from complications during hospitalisation than those that did not race again [17.3% (95% CI: 11%-24%) vs. 36.5% (95% CI: 23%-50%), p = 0.005]. Comparing pre- and post-operative racing performance, there was no significant difference in earnings per start [median £628.00, interquartile range (IQR) 115.90-1934.80 vs. £653.20, 51.00-1886.40, p = 0.7] or proportion of horses winning [51% (95% CI: 41%-61%) vs. 54% (95% CI: 44%-64%), p = 0.8] or being placed first-third [77% (95% CI: 68%-85%) vs. 71% (95% CI: 62%-80%, p = 0.5] in at least one race. MAIN LIMITATIONS: Retrospective nature of study with reliance on clinical records and public databases, limiting data available for analysis. CONCLUSIONS: Standing fracture repair is a viable treatment option for MC/MTIII or P1 fractures that returns horses to the racetrack within an acceptable time frame and is capable of restoring pre-surgical athletic ability.

Electrochemical Modification of Polypeptides at Selenocysteine.

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.

Changes in Potency and Subtype Selectivity of Bivalent NaV Toxins are Knot-Specific.

Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype NaV1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker µ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6]Sx) was shown to be the best combination as native ProTx-II potency at hNaV1.7 was conserved following ligation. At hNaV1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV1.7.

Evaluation of Peptide Ligation Strategies for the Synthesis of the Bivalent Acid-Sensing Ion Channel Inhibitor Hi1a.

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.

Synthesis and applications of mirror-image proteins.

The homochirality of biomolecules in nature, such as DNA, RNA, peptides and proteins, has played a critical role in establishing and sustaining life on Earth. This chiral bias has also given synthetic chemists the opportunity to generate molecules with inverted chirality, unlocking valuable new properties and applications. Advances in the field of chemical protein synthesis have underpinned the generation of numerous 'mirror-image' proteins (those comprised entirely of D-amino acids instead of canonical L-amino acids), which cannot be accessed using recombinant expression technologies. This Review seeks to highlight recent work on synthetic mirror-image proteins, with a focus on modern synthetic strategies that have been leveraged to access these complex biomolecules as well as their applications in protein crystallography, drug discovery and the creation of mirror-image life.

Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides.

Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

Investigation of genetic sex-specific molecular profile in well-differentiated thyroid cancer: Is there a difference between females and males?

BACKGROUND: Although more common in females, thyroid cancer is deemed to be more aggressive in males. The reasons for sex disparities in thyroid cancer are not well understood. We hypothesised that differences in molecular mutations between females and males contribute to this phenomenon. METHODS: Retrospective multicentre multinational study of thyroid nodules that underwent preoperative molecular profiling between 2015 and 2022. The clinical characteristics and mutational profiles of tumours in female and male patients were compared. Collected data included demographics, cytology results, surgical pathology, and molecular alterations. RESULTS: A total of 738 patients were included of which 571 (77.4%) were females. The extrathyroidal extension was more common in malignancies in males (chi-squared, p = 0.028). The rate of point mutations and gene fusions were similar in both sex groups (p > 0.05 for all mutations). Patients with nodules with BRAFV600E mutations were significantly younger than BRAF wild-type nodule patients (t-test, p = 0.0001). Conversely, patients with TERT promoter mutations were significantly older than patients with wild-type TERT (t-test, p < 0.0001). For patients harbouring both BRAFV600E and TERT mutations, the difference in age at presentation was significantly different in females (t-test, p = 0.009) but not in males (t-test, p = 0.433). Among females, patients with BRAFV600E and TERT mutations were significantly older than their wild-type or single-mutation counterpart (t-test, p = 0.003). CONCLUSION: The absolute rate of molecular mutations was similar in females and males. We found that extrathyroidal extension was more common in males. Moreover, BRAFV600E and TERT mutations occur at a younger age in males than in females. These two findings are factors that may explain the tendency of more aggressive disease in males.

Characteristics of PTEN Mutation in Thyroid Tumours: A Retrospective Chart Review.

While some studies suggest that PTEN mutations correlate with a low-risk phenotype in pediatric thyroid nodules, the relationship between the mutation and malignancy in the adult populations is abstruse. This study investigated whether PTEN mutations result in thyroid malignancy, and whether these malignancies are aggressive. This multicenter study involved 316 patients who underwent preoperative molecular testing, and subsequent lobectomy or total thyroidectomy at two quaternary care hospitals. A four-year retrospective review was performed on the 16 charts of patients that opted for surgery following a positive PTEN mutation on molecular testing results from January 2018 to December 2021. Of the total 16 patients, 37.5% (n = 6) had malignant tumours, 18.75% (n = 3) had non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs), and 43.75% (n = 7) had benign disease. Aggressive features were detected in 33.33% of the malignant tumours. Malignant tumours were found to have a statistically significant higher allele frequency (AF). The aggressive nodules were all poorly differentiated thyroid carcinomas (PDTCs) with copy number alterations (CNAs) and the highest AFs.

Does the likelihood of malignancy in thyroid nodules with RAS mutations increase in direct proportion with the allele frequency percentage?

BACKGROUND: Genomic testing has enhanced pre-surgical decision making for cytologically indeterminate thyroid nodules, but there remains uncertainty regarding RAS mutations. The addition of extra genetic alterations to previous driver mutation panels has been shown to improve predictive value. This study aims to evaluate the relationship between the mutant allele frequency (AF) and likelihood of malignancy in thyroid nodules with RAS mutations. METHODS: A retrospective cohort review was performed evaluating patients with indeterminate cytology (Bethesda categories III, IV and V) and ThyroSeq® v3 testing demonstrating a RAS mutation, who underwent surgery. Univariate and multivariate regression analyses were used to evaluate relationships between AF, other genetic alterations, and malignancy. RESULTS: Thirty-nine patients met criteria, 77% of the thyroid nodules (30/39) were found to be malignant. None demonstrated aggressive pathology. On univariate regression, there was no relationship between AF and likelihood of malignancy. There was, however, a significant correlation between AF and the rate of an additional genetic alteration. Multivariate analysis found a trend between RAS, a second genetic alteration and malignancy, but it did not reach statistical significance. CONCLUSIONS: There was no direct relationship between the level of allelic frequency in thyroid nodules expressing RAS mutations and the likelihood of malignancy. There was a statistically significant relationship between increasing AF and the presence of a second genetic abnormality, suggesting a possible progression from initial driver mutation and then a second genetic alteration prior to malignant transformation.

Effect of Having Concurrent Mutations on the Degree of Aggressiveness in Patients with Thyroid Cancer Positive for TERT Promoter Mutations.

This study aimed to examine whether concurrent mutations with a TERT promoter mutation are associated with a greater likelihood of more aggressive disease than a TERT promoter mutation alone. The medical records of 1477 patients who underwent thyroid surgery at two tertiary hospitals between 2017 and 2022 were reviewed. Twenty-four patients had TERT promoter mutations based on molecular profile testing. Clinicodemographic data, mutational profiles, and histopathological features were assessed. Descriptive analysis, Fisher's exact test, and binary logistic regression were performed. Seven patients had single-gene TERT promoter mutations, and 17 had concurrent mutations, including BRAF V600E, HRAS, NRAS, PIK3CA, and EIF1AX. The overall prevalence of malignancy was 95.8%, of which 78.3% were aggressive thyroid cancers. There was a statistically significant association between concurrent mutations and disease aggressiveness. The odds of having aggressive disease were 10 times higher in patients with a TERT promoter mutation and a concurrent molecular alteration than in those with a TERT promoter mutation alone. This is an important finding for thyroid specialists to consider when counseling patients concerning risk stratification and management options.

Risk Factors Associated With Reoperative Surgery for Thyroid Malignancies: A Retrospective Cohort Study.

OBJECTIVE: To examine various factors associated with an increased risk of reoperation for persistent or recurrent malignant thyroid cancers. STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary academic hospital centers. METHODS: Patients undergoing surgery for thyroid cancer at 2 tertiary academic institutions from 2006 to 2020 were included. Those who underwent a reoperative procedure were compared with patients only requiring 1 procedure. The Pearson chi-square and independent t test were used to compare group data accordingly. Furthermore, a binomial logistic regression was performed, while machine learning models were used to construct a predictive algorithm. RESULTS: This study included 2266 patients with surgically managed thyroid malignancy, of which 54 (2.4%) necessitated reoperations. Those requiring a second surgical procedure were more likely to be male (40.7% vs 20.9%, P < .001), undergo bilateral (24.1% vs 3.3%, P < .001) and lateral (16.7% vs 1.8%, P < .001) neck dissections, and have a greater number of metastatic lymph nodes (mean, 9.1 vs 3.5; P < .001) and a larger tumor size (mean, 3.0 vs 2.0 cm; P < .001). According to the binomial logistic regression model, lateral neck dissection, greater number of metastatic lymph nodes, and larger tumor size significantly increased the odds of necessitating a second procedure by 7.8 (95% CI, 2.523-24.083), 1.1 (95% CI, 1.032-1.152), and 1.3 (95% CI, 1.064-1.559), respectively. Last, machine learning models could not significantly predict the occurrence of reoperation. CONCLUSION: This study identified patient- and cancer-related characteristics associated with an increased risk of requiring reoperation for thyroid malignancies.

Outcome following emergency laparotomy in 33 UK donkeys: A retrospective multicentre study.

BACKGROUND: Emergency laparotomies in donkeys are infrequently performed and there is limited literature on the subject. OBJECTIVES: To determine findings and associated outcomes of exploratory laparotomies in donkeys. STUDY DESIGN: Descriptive retrospective study. METHODS: Donkeys undergoing emergency exploratory laparotomy for investigation and treatment of colic at seven UK referral hospitals between 2005-2017 were included. Data were retrieved from available hospital records. Descriptive statistics and inferential statistical analysis of outcomes of interest was performed in three steps. RESULTS: Thirty-three cases fulfilled the inclusion criteria. Clinical signs on presentation were available for 32 donkeys, of which 53.1% (17/32) presented for investigation of colic while in 46.9% (15/32) the presenting complaint was non-specific. Primary lesion location included small intestine (42.4%, 14/33), large colon (39.3%, 13/33), caecum (6.1%, 2/33), stomach (6.1%, 2/33) and 6.1% (2/33) had multiple abnormal findings without a clear primary lesion. Overall survival to discharge was 54.5% (18/33). Five donkeys (15.2%, 5/33) were euthanased at surgery and of those recovering from general anaesthesia a further 35.7% (10/28) were euthanased or died prior to discharge. Six donkeys (21.4%, 6/28) required a second laparotomy of which 4 (66.7%, 4/6) survived. Post-operative complications occurred in 82.1% (23/28) of cases and included hyperlipaemia (42.9%, 12/28), incisional complications (21.4%, 6/28), ileus (21.4%, 6/28) and persistent colic (17.9%, 5/28). When adjusted for other complications, donkeys with primary gastric lesions were less likely to have presented with severe colic compared with those with primary small intestinal lesions (OR: 0.07, 95% CI 0.01-0.95, p = 0.05). Only age was positively associated with death prior to discharge (OR: 1.18, 95% CI 1.03-1.36, p = 0.02). MAIN LIMITATIONS: Small sample size and retrospective design. CONCLUSION: Donkeys with abdominal lesions may present with a range of signs often not including colic. Surgical findings were diverse and survival to discharge appears to be lower than in horses.

The Impact of BRAF V600E Mutation Allele Frequency on the Histopathological Characteristics of Thyroid Cancer.

BACKGROUND: A BRAF V600E mutation in papillary thyroid cancer (PTC) has been shown to be associated with aggressive behavior. Nevertheless, not all BRAF V600E PTCs behave aggressively. Allele frequency (AF) is the number of mutated molecules divided by the total number of wild-type molecules at a specific location in the genome. The relationship between BRAF V600E AF and the histopathological features of thyroid malignancies is not well understood. We hypothesized that the BRAF V600E AF will correlate directly with aggressive histopathological behavior. The aim of this study was to examine this relationship. METHODS: A retrospective chart review was performed for patients treated for BRAF V600E thyroid malignancies from 2019 to 2022 at McGill University tertiary care hospitals (n = 317). Patients with BRAF V600E-positive malignancies that included information on AF were included (n = 44). The correlation between AF and tumor histopathological features was analyzed. RESULTS: Out of the 44 nodules with a BRAF V600E mutation, those with aggressive features of PTC had a mean AF of 25.8%, which was significantly higher than the non-aggressive group with a mean AF of 10.25% (p = 0.020). Additionally, there was a statistically significant difference in mean AF between patients with a positive sentinel LN (29%) and those with a negative sentinel LN (17.8%) (p = 0.021). Classical PTC was present in 29.5% (13/44) of nodules, with a mean AF of 15.6%. The tall cell subtype was found in 64% (28/44) of nodules, with a mean AF of 23%. Solid and hobnail subtypes were less common in this study, and there was no statistically significant relationship between AF and histopathological subtypes (p = 0.107). Nodules smaller than 1cm had a mean AF of 13.3%, while nodules ranging from 1 2cm had a mean AF of 20.6%, and those larger than 2cm had a mean AF of 27.7%. However, no statistical difference was observed between AF and nodule size (p = 0.160). CONCLUSION: In this study, BRAF V600E mutations in conjunction with AF help to determine whether thyroid malignancies will display aggressive behavior. This pre-operative finding can help thyroid specialists to determine the extent of thyroidectomy and whether lymph node dissection is required.

Prognostic Indicators of EIF1AX-Mutated Thyroid Tumor Malignancy and Cancer Aggressiveness.

The risk of malignancy (ROM) of EIF1AX-mutated thyroid nodules has been theorized to be contingent on the position of the mutation within the gene and the presence of co-existing mutations. However, due to EIF1AX's low mutation frequency, sample sizes currently reported in the literature are too diminutive to appraise the clinical utility of molecular diagnostic testing. The objective of this study was to elucidate prognostic indicators of EIF1AX-mutated thyroid tumors and cancer aggressiveness by examining a large cohort of cytologically indeterminate thyroid nodules (CITNs) that underwent molecular testing and subsequent surgical resection. This is a multicenter study involving 764 subtotal and total thyroidectomy patients that underwent preoperative molecular testing at two quaternary care hospitals. A five-year retrospective review was performed on the 42 charts of patients that opted for surgery following a positive EIF1AX mutation on ThyroseqV3 results from January 2018 to May 2022. Patient demographics, cytopathology results, molecular testing results, and postoperative histopathology were reviewed. Of the 42 surgically resected nodules that harbored an EIF1AX mutation, 16 (38.1%) were benign, six (14.3%) were non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) or well-differentiated thyroid neoplasms of uncertain malignant potential (WDT-UMPs), and 20 (47.6%) were malignant. An isolated EIF1AX mutation conferred a ROM of 47.6%, whereas the ROM for nodules with at least one additional molecular alteration was 72.7%. The ROM increased to 100% for nodules with at least one additional molecular alteration and the A113_splice site mutation. Six malignant nodules were aggressive, with five having variegated components of poorly differentiated thyroid carcinoma (PDTC). EIF1AX-mutated thyroid nodules are more susceptible to malignancy in the presence of the A113_splice site mutation and when co-mutated with RAS and/or TP53. This deleterious amalgam is associated with aggressive disease and renders these nodules PDTC. A preoperative molecular test finding of an EIF1AX mutation can be a useful tool for thyroid specialists to optimize clinical management.

Coexisting Molecular Alterations Increase the Risk of Malignancy in Thyroid Nodules with Copy Number Alterations.

Molecular mutations and alterations play a role in thyroid tumorigenesis. Different alterations are associated with different clinical and pathological characteristics. Copy number alterations (CNAs) are known to be present in some thyroid tumors; however, their idiosyncratic clinicopathological implications are not yet well elucidated. A retrospective chart review was performed to identify patients with CNAs on pre-operative molecular testing results who subsequently underwent surgical treatment between January 2016 and April 2022 at McGill University teaching hospitals. Of the 316 patients with thyroid nodules who opted for molecular testing with ThyroSeqV3 followed by surgery, 67 (21.2%) nodules were positive for CNAs, including 23 Bethesda III, 31 Bethesda IV, 12 Bethesda V and 1 Bethesda VI nodules. On surgical pathology, 29.9% were benign and 70.1% were malignant or non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Among those that were malignant/NIFTP, 17.02% were considered to be aggressive cancers. The presence of other molecular alterations was found to be an independent predictor of malignancy in multivariate analysis (OR = 5.087, 95% C.I. = 1.12-23.04, p = 0.035). No unique factor was correlated with aggressiveness; however, CNA-positive thyroid nodules that were associated with high-risk mutations such as BRAF V600E, TP53, NTRK1/3 fusion, or PTEN mutation with high allele frequency (AF) ended up being aggressive cancers. Most of the CNA-positive thyroid nodules resulted in follicular patterned tumors in 41 (65.2%) cases and oncocytic tumors in 20 (29.9%) cases. This study demonstrates that 70.1% of surgically resected thyroid nodules with CNAs were malignant/NIFTP. Most CNA-positive thyroid nodules were either oncocytic patterned tumors or follicular patterned tumors. Furthermore, CNA-positive thyroid nodules were more likely to be malignant if they were associated with other molecular alterations or mutations.