This case involves a boy with progressive swelling of the neck diagnosed as Cystic Hygroma by ultrasound and MRI. After an initially wait-and-see approach because of his age and lack of symptoms, increased swelling led to surgical excision, which is preferred over sclerotherapy. After 2 months there was minimal scarring and no recurrence.

Objectives: To assess the efficacy of magnetic resonance (MR) lymphography with gadobutrol contrast for sentinel lymph node (SLN) mapping in early-stage oral squamous cell carcinoma (OSCC). Methods: This pilot study compared the identification of SLNs by MR lymphography using a gadolinium-based contrast agent (gadobutrol) to conventional [99mTc]Tc-nanocolloid lymphoscintigraphy (including single-photon emission computed tomography/computed tomography (SPECT/CT)) in 10 early-stage OSCC patients undergoing SLN biopsy. The patients initially underwent conventional lymphoscintigraphy following the peritumoral administration of indocyanine green [99mTc]Tc-nanocolloid (120 megabecquerel; ~0.5 mL). Subsequently, 0.5–1.0 mL gadobutrol was peritumorally injected, and MR imaging was acquired for 30 min. The following day, the identified SLNs were harvested and subjected to a histopathological assessment. The MR lymphography and [99mTc]Tc-nanocolloid lymphoscintigraphy results were evaluated and compared with respect to those of the SLN identification. The reference standard consisted of a histopathological evaluation of the harvested SLNs, complementary neck dissection specimens, and follow-up data. Results: The MR lymphography detected 16 out of 27 SLNs identified by [99mTc]Tc-nanocolloid lymphoscintigraphy, revealing an additional SLN that did not harbor metastasis. MR lymphography failed to identify any SLNs in one patient. Of the seven histopathologically positive SLNs detected by [99mTc]Tc-nanocolloid lymphoscintigraphy, three were identified by MR lymphography. All patients remained disease-free after a median follow-up of 16 months. Compared to [99mTc]Tc-nanocolloid lymphoscintigraphy, MR lymphography using gadobutrol achieved an SLN identification rate of 59%, a sensitivity of 75%, and a negative predictive value of 86%. Conclusions: MR lymphography using gadobutrol demonstrates limited reliability for SLN mapping in early-stage OSCC.

Objectives: To assess whether there are differences in the effects of time to treatment interval (TTI) on patient survival for head and neck cancer (HNC) sites in order to provide evidence that can support decision-making regarding prioritizing treatment. Materials and methods: Patients in the Netherlands with a first primary HNC without distant metastasis between 2010 and 2014 were included for analysis (N =10,486). TTI was defined as the time from pathologic diagnosis to the start of initial treatment. Overall survival (OS), cox regression analyses and cubic spline hazard models were calculated and visualized. Results: Overall, the hazard of dying was higher (HR = 1.003; 95 % CI 1.001-1.005) with each additional day until treatment initiation. The pattern, as visualized in cubic spline graphs, differed by site the hazard increased more steeply with increasing TTI for oral cavity cancer. For oropharyngeal and laryngeal cancer, a slight increase commenced after a longer TTI than for oral cavity cancer, while there was hardly an increase in hazard with increasing TTI for hypopharyngeal cancer. Conclusion: The relationship between longer TTI and decreased survival was confirmed, but slight variations in the pattern of the hazard of dying by TTI by tumour site were observed. These findings could support decisions on prioritizing treatment. However, other aspects such as extent of treatment and quality of life should be investigated further so this can also be included.

Objectives. Diagnosing adenoid cystic carcinoma (AdCC) is challenging due to histopathological variability and similarities with other tumors. In AdCC pathogenesis, the cellular myeloblastosis gene (c-MYB) often exhibits a MYB::NFIB fusion from a reciprocal translocation. This study aimed to assess the predictive accuracy of MYB immunohistochemistry for detecting this translocation compared to fluorescence in situ hybridization (FISH). Study design. This study included 110 AdCC patients (1999-2017) from two Dutch head and neck centers using tissue microarrays and full slides. Median MYB expression levels by immunohistochemistry were compared based on translocation status by FISH, and differences within clinicopathological parameters were examined. An immunohistochemical cut-off was established to estimate the translocation. Results. MYB immunohistochemistry was available in 90/110 patients, with a median expression of 27%. FISH was interpretable in 79/108 tumors, identifying MYB::NFIB fusion in 44 (56%). Among 62 patients with both MYB expression and translocation data, the fusion was present in 38 (61%). These tumors had higher MYB expression (30%) than nontranslocated tumors (6%); P = .02. A 60% MYB expression cut-off yielded 100% specificity for detecting the translocation but had no prognostic value. Conclusions. Although MYB protein expression alone lacks diagnostic precision, protein expression > 60% predicted the MYB:: NFIB fusion in all tumors. (Oral Surg Oral Med Oral Pathol Oral Radiol 2024;138:772-780)

Besides genetic alterations, the cellular environment also determines disease onset and progression. When different cell types contribute to disease outcome, this imposes environmental challenges as different cell types likely differ in their extracellular dependencies. Hsa-microRNA-31-5p (miR-31) is highly expressed in keratinocytes of psoriatic skin, and we show that expression in keratinocytes is induced by limited glucose availability and enables increased survival under limiting glucose conditions by increasing glutamine metabolism. In addition, miR-31 expression results in not only secretion of specific metabolites (aspartate and glutamate) but also secretion of immunomodulatory factors. We show that this miR-31-induced secretory phenotype is sufficient to induce Th17 cell differentiation, a hallmark of psoriasis. Inhibitors of miR31-induced metabolic rewiring and metabolic crosstalk with immune cells alleviate psoriasis pathology in a mouse model of psoriasis. Together our data illustrate an emerging concept of metabolic interaction across cell compartments that characterizes disease development, which can be employed to design effective treatment options for disease, as shown here for psoriasis.

FGFR1 protein expression was determined by immunohistochemistry on tissue microarray slides containing 443 head and neck squamous cell carcinoma. FGFR1 gene copy-numbers were determined by fluorescence in situ hybridization with FGFR1 FISH probes. FGFR1: fibroblast growth factor receptor 1, HPV: human papillomavirus

The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells towards osteoblast and myocyte phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation. For these studies pre-adipocytes isolated from mouse adipose tissue and the 3T3-L1 (pre)adipocyte cell line were applied. We performed FHL2 gain of function and knockdown exper-iments followed by extensive RNAseq analyses and phenotypic characterization of the cells by oil-red O (ORO) lipid staining. Through affinity-purification mass spectrometry (AP-MS) novel FHL2 interacting proteins were identified. Here we report that FHL2 is expressed in pre-adipocytes and for accurate adipocyte differentiation, this protein needs to be downregulated during the early stages of adipogenesis. More specifically, constitutive overexpression of FHL2 drastically inhibits adipocyte differentiation in 3T3-L1 cells, which was demonstrated by suppressed activation of the adipogenic gene expression program as shown by RNAseq analyses, and diminished lipid accumulation. Analysis of the protein-protein interactions mediating this repressive activity of FHL2 on adipogenesis revealed the interaction of FHL2 with the Nuclear factor of activated T-cells 5 (NFAT5). NFAT5 is an established inhibitor of adipocyte differentiation and its knockdown rescued the inhibitory effect of FHL2 overexpression on 3T3-L1 differentiation, indicating that these proteins act cooperatively. We present a new regulatory function of FHL2 in early adipocyte differentiation and revealed that FHL2-mediated inhibition of pre-adipocyte differentiation is dependent on its interaction with NFAT5. FHL2 expression increases with aging, which may affect mesenchymal stem cell differentiation, more specifically inhibit adipocyte differentiation.

Purpose Evaluating the Crystal Cam handheld gamma-camera for preoperative and intraoperative sentinel lymph node (SLN) localization in early-stage oral cancer. Methods The handheld gamma-camera was used complementary to conventional gamma-probe guidance for intraoperative SLN localization in 53 early-stage oral cancer patients undergoing SLN biopsy. In 36 of these patients, a blinded comparison was made between preoperative handheld gamma-camera and lymphoscintigraphy outcomes. Of those, the reliability for marking the SLN’s location using both handheld gamma-camera and a 57 Co-penpoint marker was evaluated in 15 patients. Results In the entire cohort, the handheld gamma-camera preoperatively detected 116/122 (95%) of SLNs identified by lymphoscintigraphy. In those patients where the observer was blinded for lymphoscintigraphy ( n = 36), 71/77 (92%) SLNs were correctly identified by handheld gamma-camera. Overlooked SLNs by handheld gamma-camera were mainly located near the injection site. The SLN’s marked location by handheld gamma-camera and 57 Co-penpoint marker was considered accurate in 42/43 (98%) SLNs. The intraoperative use of the handheld gamma-camera led to the extirpation of 16 additional ‘hot’ lymph nodes in 14 patients, 4 of which harbored metastases, and prevented 2 patients (4%) from being erroneously staged negative for nodal metastasis. In those with follow-up ≥ 24 months or false-negative outcomes < 24 months following SLNB, a sensitivity of 82% and negative predictive value of 93% was obtained. Conclusion The Crystal Cam handheld gamma-camera offers reliable preoperative and intraoperative SLN localization and might reduce the risk of missing a malignant SLN during surgery. Detecting SLNs near the injection site by handheld gamma-camera remains challenging.

(A) FGFR1 protein overexpression and low FGFR1 expression in HPV-positive and HPV-negative head and neck squamous cell carcinoma patients. FGFR1 protein was overexpressed in 82% of HPV-positive and 75% of HPV-negative head and neck squamous cell carcinoma. (B) FGFR1 gene copy-numbers in HPV-positive and HPV-negative head and neck squamous cell carcinoma patients. 3% of FGFR1-overexpressing HPV-negative head and neck squamous cell carcinoma had FGFR1 amplification, 5% had a copy-number gain and 92% had normal gene copy-numbers. All FGFR1-overexpressing HPV-positive head and neck squamous cell carcinoma had normal FGFR1 gene copy-numbers. (C) The Kaplan-Meier overall survival curve for FGFR1 protein overexpression shows no relationship with survival for HPV-positive head and neck squamous cell carcinoma patients (FGFR1 overexpression: 7/33 events, low FGFR1 expression: 2/8 events). Overall survival time was missing for one patient, tissue microarray cores were unavailable for one tumor and two patients were excluded from survival analysis because they were treated with palliative intent. (D) The Kaplan-Meier disease-free survival curve also shows no relationship with survival for HPV-positive head and neck squamous cell carcinoma (FGFR1 overexpression: 10/33 events, low FGFR1 expression: 1/7 events). Disease-free survival time was missing for two patients, tissue microarray cores were unavailable for one tumor and two patients were excluded from survival analysis because they were treated with palliative intent.

Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a col-lective term used for multiple tumor types including squamous cell car-cinomas and salivary gland adenocarcinomas. Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo-and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clin-ical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio -sensi-tizing potential of cisplatin and carboplatin could be validated. How-ever, cetuximab conveyed radioprotection in most models. HNC -tar-geted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. Conclusions: Organoids hold potential as a diagnostic tool in personal-ized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation.

OBJECTIVE:In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS:We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS:Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS:Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

We reported a case series of thin (n = 32) and ultrathin (n = 4) submental artery perforator flaps (SMAPF) for precise reconstruction following T1-2 oral cavity cancer resection. The former is indicated for tongue reconstruction involving the tongue tip, and buccal lining replacement involving the mouth corner, while the latter is specifically tailored to restore superficial buccal defects after resection of small early cancer originating from oral submucous fibrosis. All flaps survived. Most flap reconstructions reached satisfactory cosmetic results. Based on this series, we discussed the indications of thin and ultrathin SMAPFs in intaoral reconstruction, and surgical tips for flap thinning.

Objectives: Surgical removal of squamous cell carcinoma of the tongue (SCCT) with tumour-free margin status (>= 5 mm) is essential for loco-regional control. Inadequate margins (< 5 mm) often indicate adjuvant treatment, which results in increased morbidity. Ultrasound (US)-guided SCCT resection may be a useful technique to achieve more adequate resection margins compared to conventional surgery. This study evaluates the application and accuracy of this technique. Methods: Forty patients with SCCT were included in a consecutive US cohort. During surgery, the surgeon aimed for a 10-mm echographic resection margin, while the tumour border and resection plane were captured in one image. Ex-vivo US measurements of the resection specimen determined whether there was a need for an im-mediate re-resection. The margin status and the administration of adjuvant treatment were compared those of with a consecutive cohort of 96 tongue cancer patients who had undergone conventional surgery. A receiver operating characteristic analysis was done to assess the optimal margin of ex-vivo US measurements to detect histopathologically inadequate margins. Results: In the US cohort, the frequency of free margin status was higher than in the conventional cohort (55% vs. 16%, p < 0.001), and the frequency of positive margins status (< 1 mm) was lower (5% vs. 15%, respectively, p < 0.001). Adjuvant radiotherapy was halved (10% vs. 21%), and the need for re-resection was comparable (10% vs. 9%). A cut-off value of 8 mm for ex-vivo measurements prevented histopathologically inadequate margins in 76%. Conclusion: US-guided SCCT resections improve margin status and reduce the frequency of adjuvant radiotherapy.

Psoriasis is characterized by a combination of keratinocyte hyperproliferation and immune cell activation. Immune cell activation requires increased glucose consumption, consequently limiting glucose availability for other cell types like keratinocytes. In psoriasis Hsa-microRNA-31-5p (miR-31) is highly expressed in keratinocytes. Here we show that miR-31 expression in keratinocytes is induced by limited glucose availability and increases survival under limiting glucose conditions, by increasing glutamine metabolism. In addition, miR-31 induced glutamine metabolism results in secretion of specific metabolites (aspartate and glutamate) but also immuno-modulatory factors. We show that this miR-31-induced secretory phenotype is sufficient to induce Th17 cell differentiation, a hallmark of psoriasis. Inhibition of glutaminase (GLS) using CB-839 impedes miR31-induced metabolic rewiring and secretion of immuno-modulatory factors. Concordantly, pharmacological targeting of GLS alleviated psoriasis pathology in a mouse model of psoriasis. Together our data illustrate an emerging concept of metabolic interaction across cell compartments that characterizes disease development, which can be employed to design effective treatment options for disease, as shown here for psoriasis.

Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9–mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer.

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Evaluation of relevant clinical outcomes in patients with bilateral cleft lip and palate (BCLP) after secondary aveolar bone grafting (SABG) and premaxilla osteotomy (PMO), through the use of a new scoring system. Data were collected retrospectively from all patients with BCLP who were operated on between 2004 and 2014, at the end of follow-up. The treatment protocol consisted of SABG + PMO in patients aged between 9 and 13 years. At the end of follow-up, the following parameters were scored: (un)interrupted dental arch, skeletal sagittal relationship, bone height using the Bergland/Abyholm criteria, and the presence of postoperative fistula. These parameters were combined to produce a dento-maxillary scoring system, giving a final score between 1 and 10. For statistical analysis, the independent t-test was used. Of 55 children, 45 were suitable for analysis. The mean age at time of surgery was 12.0 years (8.9 -16.4 yrs), and the mean follow-up time was 11.7 years (5.8-15.8 yrs). The average number of surgeries executed under general anesthesia was 6 (range: 3-11). The average dento-maxillary score in this patient cohort was 7.6 (1-10; median: 8). Among these patients, 31 had an uninterrupted dental arch; the average Bergland/Abyholm score was 2.07; 30 patients exhibited an Angle class I incisor relationship; and, in 38 cases, the oronasal communication was closed after SABG + PMO treatment. A significant effect of fistulas was seen on dento-maxillary score (p = 0.001). Specifically, a significant effect of fistulas was seen on interrupted dental arch (p = 0.002) and on Bergland/Abyholm score (p = 0.037). The proposed dento-maxillary scoring system is a straightforward tool that can be used to describe and analyze the amount of dento-maxillary rehabilitation at the end of the treatment. Persistence of oronasal fistulas in patients with BCLP has a significant impact on interruption of the dental arch, and can influence dental results at the end of the second decade. (C) 2020 The Author(s). Published by Elsevier Ltd on behalf of European Association for Cranio-MaxilloFacial Surgery.