Abstract Excessive centrosome activation is detrimental to normal cells by hampering bipolar spindle formation during mitosis and inducing cell death. In the highly aggressive anaplastic thyroid cancer (ATC), cells expressing the aldehyde dehydrogenase–positive (ALDH+) stem cell trait are enriched in supernumerary centrosomes and tolerate centrosome amplification, which offers these cells a growth advantage. Our earlier work characterized the chromosomal instability (CIN) within this cell population, which was due to dysfunctional centrosomes manifesting as a deficiency in pericentriolar material. Here, we report a novel 2-4-morpholinoanilino-6-[(2-exo-norbornyl) amino]-purine (MEAP) that preferentially restores centrosome microtubule nucleation activity in centrosome-deficient ALDH+ ATC cells. Exposure of these cells to MEAP induced pericentriolar accumulation and microtubule nucleation activity of supernumerary centrosomes, resulting in spindle multipolarity. Consequently, MEAP was capable of preferentially eliminating ALDH+ ATC cell population, thus reducing cell spherogenesis and self-renewal capacity. In a preclinical ATC mouse model, MEAP preferentially eliminated ALDH+ cell clusters, increased the rate of spindle multipolarity, decreased CIN, and generated a robust antitumor response. Lastly, we identified that the selective activity of MEAP in ALDH+ cells involved the modulation of NEDD9–STAT3 signaling. Together, these findings support the potential of targeting centrosome amplification as an alternative therapeutic approach for aggressive ALDH+ cancers failing first-line therapeutics. Significance: This study identifies MEAP as the first selective vulnerability of ALDH+ ATC stem cells, their dysfunctional supernumerary centrosomes. Unlike broad chemotherapeutics, MEAP exploits this cancer-specific centrosome amplification by paradoxically hyperactivating them via NEDD9–STAT3 disruption, forcing multipolar mitosis and ALDH+ elimination while sparing bulk tumor cells. Validates centrosome declustering as a novel therapeutic axis for aggressive, stem-like cancers failing standard therapies, with robust in vivo efficacy and a mechanistic framework (NEDD9–STAT3–centrosome axis) for precision translation.

MEAP induces PCM accumulation at inactivate centrosomes during interphase. A, Representative images of centrin-1 (centriole marker) and PCNT costaining in THJ-16T cells with a normal number of centrosomes. In DMSO-treated cells, one of the two centrosomes often displays reduced PCNT staining during interphase, whereas MEAP-treated cells show more uniform PCNT accumulation across both centrosomes. Cell-cycle stage was inferred based on centriole duplication status (before duplication in first and last columns) and the distance between centrosomes, ensuring equivalent comparisons between DMSO- and MEAP-treated cells. B, Quantification of PCNT fluorescence intensity at individual centrosomes reveals that MEAP primarily increases PCNT levels at the less active (“weaker”) centrosome of the pair. Centrosome identity was determined by intercentrosomal distance during interphase. Quantification was performed in Fiji/ImageJ by drawing a 2.5-μm line through the center of one or both centrosomes and using the “Plot Profile” function to calculate integrated intensity after background subtraction. Data represent three biological replicates, with 25 cells scored per condition per replicate (n = 3). Data representation (mean ± SD) *, P < 0.01 by one-way ANOVA with Tukey post hoc test (weak DMSO vs. weak MEAP). C, Representative images of centrin-1 and PCNT costaining in THJ-11T and THJ-16T cells with supernumerary centrosomes. DMSO-treated cells show frequent PCNT-deficient centrioles, whereas MEAP-treated cells exhibit widespread PCNT acquisition. D, Quantification of the percentage of centrioles with visible PCNT accumulation. MEAP significantly increases the proportion of PCM-positive centrioles in both cell lines. At least 25 cells with centrosome amplification were analyzed per condition across three biological replicates (n = 3). Data representation (mean ± SD) **, P < 0.01; ***, P < 0.001 by t test with Welch correction.

Importance:A unified salivary gland carcinoma (SGC)-specific tumor-node-metastasis (TNM) classification can enhance prognostic accuracy, support clinical decision-making, and improve the quality of patient care. Objective:To derive and validate an SGC-specific pTNM classification with improved prognostic accuracy and optimized stage distribution for version nine of the American Joint Committee on Cancer/Union for International Cancer Control staging protocol. Design, Setting, and Participants:This retrospective prognostic cohort study derived a novel pTNM classification using data from the National Cancer Database (NCDB) of patients with surgically treated major SGC (2012-2017) and validated it in an international major SGC cohort (2008-2021) and a single-institution minor SGC cohort (Memorial Sloan Kettering Cancer Center; 1985-2016). Data were analyzed from June to November 2024. Exposures:Surgery with or without postoperative radiotherapy or chemoradiotherapy. Main Outcomes and Measures:The primary end point was overall survival (OS). Cox proportional hazards multivariable analysis was used to confirm the prognostic importance of pathologically positive lymph node (LN) number and extranodal extension (pENE) and derive an optimal pTNM classification. Results:The NCDB dataset included 8409 patients with SGC: 7659 with M0 disease (5748 with pN0 disease and 1911 with pN+ disease) and 750 with M1 disease. Among the 7659 patients with M0 disease, the median (IQR) age was 60 (48-71) years, and 3861 (50.4%) were male. The median (IQR) follow-up was 88.4 (72.3-108.5) months. The 5-year OS was 87.2% (95% CI, 86.3-88.0) for N0 disease, 68.2% (95% CI, 63.9-72.8) for 1 positive LN without pENE, 60.2% (95% CI, 53.5-67.5) for 2 positive LNs without pENE, 68.4% (95% CI, 58.0-76.6) for 3 positive LNs without pENE, 47.5% (95% CI, 41.6-52.8) for more than 3 positive LNs without pENE, and 41.4% (38.1-44.8) for pENE-positive LNs. Multivariable analysis confirmed the independent prognostication of LN count compared with pN0 disease (1 positive LN: adjusted hazard ratio [aHR], 1.70; 95% CI, 1.44-2.01; 2 positive LNs: aHR, 1.61; 95% CI, 1.31-1.98; 3 positive LNs: aHR, 2.10; 95% CI, 1.65-2.68; 4 positive LNs : aHR, 2.46; 95% CI, 1.87-3.24; more than 4 positive LNs: aHR, 2.07; 95% CI, 2.08-2.91) and pENE-positive LNs compared with pENE-negative LNs (aHR, 1.27; 95% CI, 1.10-1.48). The proposed pN classification were pN1 for 1 to 3 positive LNs and pENE negativity and pN2 for more than 3 positive LNs or pENE positivity. Model fit improved with the proposed pN classification vs the current pN classification (Akaike Information Criterion, 26 442 vs 26 483). Based on the aHR model, the following stage groups were proposed: stage I: T1N0 (1 [reference]); stage II: T2N0 (aHR, 1.34; 95% CI, 1.11-1.61); stage IIIA: T1-2N1 or T3-4N0 (aHR, 2.36; 95% CI, 1.99-2.80); stage IIIB: T1-2N2 or T3-4N1-2 (aHR, 5.15; 95% CI, 4.38-6.06); and stage IV: M1 disease (aHR, 13.61; 95% CI, 11.37-16.29). The C index values were similar (proposed classification: 0.792; current classification: 0.790), while the AIC improved slightly (proposed classification: 26 441; current classification: 26 482). Stage-specific OS differences were evident in both the international major SGC cohort (n = 1015) and Memorial Sloan Kettering Cancer Center minor SGC cohort (n = 444). Conclusions and Relevance:This unified, SGC-specific staging system improved prognostic accuracy and sample size balance and was applicable to both major and minor SGCs.

MEAP preferentially induces multipolar spindles and G2/M arrest in ALDH+ ATC cells. A, Representative image of “inactive” centrosomes observed in ALDH+ cells at mitosis, defined as centrosomes which are visibly devoid of spindle-forming capacity. Cell model represented is THJ-11T. B, Representative images of a pseudobipolar and multipolar spindle in THJ-16T cells with supernumerary centrosomes. C, Quantification of % inactivated centrosomes during mitosis. Sorted cells were treated with DMSO or 300 nmol/L MEAP for 48 hours. D, Quantification of the ratio of cells with supernumerary centrosomes (>2 centrosomes). Sorted cells were treated with DMSO or 300 nmol/L MEAP for 48 hours. E, Quantification of spindle configuration in ALDH+ cells treated with DMSO or MEAP. Pseudobipolar spindles are defined as >2 centrosomes assembled into two spindle poles, and multipolar spindles were defined as >2 centrosomes assembled into >2 spindle poles (represented in B). Spindle multipolarity THJ-11T/16T (MEAP): ALDH+ 32 ± 5% vs. 8% ± 3% ALDH− (P < 0.001, unpaired t test, n = 3), and no change ALDH− (P = 0.42). F, Impact of MEAP on centrosome fragmentation (defined as % of PCNT-positive structures lacking centrin-1). G, Representative flow cytometry panel and quantification of BrdU/DAPI cell-cycle compartmentalization assay of sorted THJ-16T cells treated with 300 nmol/L MEAP or DMSO. Treated cells were harvested at the indicated time points. n = 3 biological replicates. H, Quantification of propidium iodide (PI)/Annexin V apoptosis assay of sorted THJ-16T cells treated with MEAP. n = 3 biological replicates. I, Quantification of β-galactosidase senescence assay of sorted THJ-16T cells treated with MEAP. n = 3 biological replicates. Scale bars, 10 μm. Data representation (mean ± SD) **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey post hoc test.

MEAP selectively eliminates ALDH+ ATC cells and attenuates spherogenesis and tumorigenic potential. A, Quantification of the impact of small molecules (300 nmol/L) on the ratio (%) of cells expressing the cancer stem-like cell marker ALDH+ in three ATC models. B, Viability assay illustrating that MEAP’s selectivity against ALDH+ THJ-16T cells are due to differential effects on ALDH+ vs. ALDH− cell growth. C, Dose-responsive inhibition of the %ALDH+ cell population in THJ-16T ATC cells after 72 hours treatment of MEAP, in which the addition of the ALDH enzymatic inhibitor “DEAB” is used to establish background levels. D, Impact of MEAP on ALDH enzymatic assay. Propanal is added to a cell mixture together with MEAP, DEAB, or DMSO. Reaction inhibition is measured by total NAD+ reduction in MEAP- or DEAB-treated (positive control) samples compared with those treated with DMSO (negative control); decreasing dose of substrate is for assessing competitive inhibition (n = 3). E, Representative image and quantification of the impact of MEAP on tumorsphere formation in several ATC cell lines. A total of 1,000 cells were seeded in polyhema-coated plates in tumorsphere growing media (DMEM/F12 + EGF + B27). Images were taken after 7 days, using 10× magnification; for quantification, brightfield microscopy was used to count spheres more than 50 μm. Quantification is normalized to DMSO-treated for each cell line. F, Limiting dilution transplantation assay to measure the impact of MEAP pretreatment on ATC cell tumor seeding potential in vivo. THJ-16T cells were recovered for 5 days after pretreatment and injected at 5 × 105, 1 × 105, and 0.5 × 105 cells per mouse (n = 5 per dose) into the right flank of NOD-SCID mice. Tumor growth was assessed by palpitation and confirmed at necropsy after 35 days. ELDA stem cell frequencies were DMSO 1/262,000 vs. MEAP 1/1,310k (P = 0.015); equivalent to ∼80% drop in CIC number. Data representation (mean ± SD) *, P < 0.01 by one-way ANOVA with Tukey post hoc test.

OBJECTIVES:To describe metastatic characteristics and outcomes in patients with de novo metastatic (cM1) head and neck squamous cell carcinoma (HNSCC). METHODS:cM1 HNSCC patients diagnosed between 2001 and 2024 were reviewed. Metastatic characteristics and management patterns were collected, including the timing of locoregional radiotherapy (LR-RT) relative to first-line systemic therapy. Overall survival (OS) was estimated using the Kaplan-Meier method, and multivariable Cox regression analyses (MVA) identified prognostic factors. RESULTS:Among 10,273 newly diagnosed HNSCC cases, 222 (2.2%) had cM1 disease (HPV-associated: 48/2593 [1.9%]; HPV-independent: 174/7680 [2.3%]). cM1 characteristics were similar between HPV-associated and HPV-independent disease, including oligometastatic presentation (1-5 lesions; 31% vs 43%, p = 0.21) and frequency of lung-only metastases (83% vs 77%, p = 0.46), although mediastinal involvement was more common in HPV-associated disease (40% vs 23%, p = 0.034). LR-RT was delivered to 191 (86%) patients: 127 (57%) as sole treatment, 55 (25%) before, and 9 (4%) after first-line systemic therapy. After median follow-up of 25 months, median OS was 7 months (6.0-8.8). On MVA, LR-RT (HR 0.49 [0.32-0.75]), HPV-associated disease (HR 0.62 [0.43-0.88]), ECOG 0-1 (HR 0.63 [0.47-0.86]), first-line immunotherapy (HR 0.52 [0.33-0.82]), and lung-only metastases (HR 0.67 [0.48-0.95]) were associated with longer OS. Among patients receiving LR-RT, a radiation dose ≥ 50 Gy was associated with improved OS (HR 0.53 [0.34-0.83]). CONCLUSIONS:cM1 HNSCC has a poor prognosis, highlighting a substantial unmet need. First-line immunotherapy and LR-RT, particularly with ≥ 50 Gy, were associated with improved OS and warrant prospective evaluation.

Impact of analogues on the colony forming abilities and proliferation of anaplastic thyroid carcinoma cells.

OBJECTIVES:To evaluate the prognostic importance of impaired vocal cord mobility (VCM) in T2N0 glottic cancer. METHODS:All patients with T2N0 glottic cancer treated with partial laryngeal IMRT in 2009-2021 in our institution were retrospectively reviewed. For comparison, cohorts with T1N0 and T3N0 disease were also included. Locoregional failure (LRF), disease-free survival (DFS), and overall survival (OS) were compared among T1N0, T2N0 with normal VCM (T2-Normal-VCM), T2N0 with impaired VCM (T2-Impaired-VCM), and T3N0 groups. Multivariable analysis (MVA) assessed the prognostic value of VCM within the T2N0 group. RESULTS:A total of 642 cases were included: 288 T1N0, 224 T2N0 (147 T2-Normal-VCM, 77 T2-Impaired-VCM), and 130 T3N0. Median follow-up was 5.0 years (IQR 3.4-6.2). Five-year LRF for T1N0, T2-Normal-VCM, T2-Impaired-VCM, and T3N0 were 4% (95% CI 2-6), 9% (5-15), 27% (17-38), and 35% (27-44), respectively. Corresponding DFS was 83% (78-88), 80% (73-87), 55% (45-68), and 50% (41-60), while OS was 85% (80-90), 86% (80-92), 71% (61-83), and 59% (50-69), respectively. In T2N0, MVA confirmed that impaired VCM was associated with higher LRF (aHR 3.72 [95% CI 1.79-7.71], p < 0.001), lower DFS (aHR 2.74 [1.68-4.45], p < 0.001), and lower OS (aHR 2.07 [1.17-3.67], p = 0.013). CONCLUSIONS:In this contemporary cohort, LRF rates increased stepwise from T1N0 to T2-Normal-VCM, T2-Impaired-VCM, and T3N0 glottic cancer. Within T2N0 disease, impaired VCM is an adverse prognostic factor, supporting subdivision into T2a (normal VCM) and T2b (impaired VCM) in future TNM revisions. Prospective studies are warranted to assess whether treatment intensification can improve outcomes for T2-Impaired-VCM disease. LEVEL OF EVIDENCE: 3:

MEAP causes multipolar spindles and mitotic failure in ALDH+ cells harboring centrosome amplification and leads to reduced missegregated chromosomes in a NEDD9/STAT3-dependent manner. A, Western blot illustrating NEDD9 knockdown in combination with STAT3 or STAT3-CA (constitutively activated mutant with A662C, N664C point mutations) expression. B, MT regrowth assay illustrating that STAT3-CA but not STAT3 overexpression can abrogate MEAP-induced centrosome MT-nucleation. At least 25 centrosomes were scored per experiment, n = 3 biological replicates. C, MEAP induced γ-tubulin accumulation was inhibited by ectopic expression of constitutively activated STAT3 (STAT3-CA). Left, representative images and quantification of γ-tubulin levels in DMSO- or MEAP-treated (100 nmol/L, 24 hours) control or STAT3-CA–overexpressing ALDH+ THJ-16T cells. Right, line graph panel indicating the intensity of centrosome pairs or individual centrosomes of cells, measured using the “plot profile” function of Fiji/ImageJ software. The region quantified is depicted by a white line in the left. D, Quantification of the relative rate of bipolar, pseudobipolar (defined as >2 centrosomes assembled into two spindle poles), or multipolar (defined as >2 centrosomes assembled into >2 spindle poles) centrosomes in the different groups. E, Representative image (original magnification 63×) and quantification of the impact of MEAP on rate of missegregated chromosomes (defined by presence of lagging chromosomes or chromosome bridges). Data representation (mean ± SD) *, P < 0.05; ***, P < 0.001 by one-way ANOVA with Tukey post hoc test. F, Schematic of a model which integrates the impact on centrosome/spindle and chromosomal missegregation observed following MEAP treatment. Scale bars, 10 μm.

Lymphedema is a chronic complication of breast cancer treatment, and early intervention is crucial to reduce morbidity. This study evaluated the role of blood-based cytokine biomarkers in the prognostication of breast cancer-related lymphedema (BCRL) to improve risk prediction. A secondary analysis of inflammatory biomarkers for BCRL was performed using a previously published cohort of 147 patients with breast cancer who had undergone serum cytokine profiling during their treatment at the Princess Margaret Cancer Centre from 2010 to 2014. Prognostic cytokine variables for lymphedema were selected by regression analysis and independence from known clinical risk factors. Regression-based modeling was employed to integrate prognostic variables for the prediction of lymphedema occurrence. We identified the immunostimulatory cytokine IFNα2A as a potential biomarker for lymphedema development [OR, 3.10; 95% confidence interval (CI), 1.05-9.51; P = 0.042], independent from known clinical risk factors. Furthermore, Kaplan-Meier analysis demonstrated 3-year lymphedema-free survival of 95% (90%-100%) versus 85% (77%-94%) for below versus above median concentrations of IFNα2A (P = 0.026). In combination with an established clinical risk regression-based model, patients identified as high risk based on clinical factors alone were able to be correctly reclassified as low risk by IFNα2A in 31% (eight of 26) of cases. Our combined logistic regression model using both IFNα2A and clinical risk score achieved an AUC of 0.895 (95% CI, 0.796-0.971) and Brier score of 0.101 (95% CI, 0.061-0.149), representing a favorable improvement compared with the logistic regression model using clinical risk factors alone. IFNα2A in combination with established clinical risk factors may be useful for improving BCRL prognostication. SIGNIFICANCE:The cytokine IFNα2A was identified as a potentially complementary biomarker to improve the stratification of high- and low-risk patients for BCRL. This will help enable earlier intervention to reduce long-term morbidity for those at high risk for lymphedema while minimizing burdensome interventions for those at low risk.

OBJECTIVES:To report disease-free survival (DFS) in p16-positive non-oropharyngeal head-and-neck squamous-cell carcinoma (HNSCC). METHODS:Curatively-treated non-oropharyngeal HNSCC (2009-2021) were reviewed. DFS was compared among p16-positive, p16-negative, and p16-untested cases for the overall cohort and by disease site. Multivariable analysis estimated adjusted hazard ratio (aHR) for p16-positivity. RESULTS:Among 3061 non-oropharyngeal HNSCC, 91 p16+, 375 p16-, 2595 untested; median follow-up 4.7 years. P16-positive patients had superior 5-year DFS versus p16-/untested (74% vs. 48%/59%; aHR 0.40 [0.26-0.61], p < 0.001). This advantage was evident in the larynx (86% vs. 52%/63%; aHR 0.26 [0.09-0.71], p = 0.005), hypopharynx (70% vs. 25%/37%; aHR 0.55 [0.10-0.50], p < 0.001), and nasal cavity (82% vs. 38%/66%; aHR 0.31 [0.11-0.88], p = 0.028). No significant DFS differences were seen in paranasal sinus (40% vs. 64%/41%; p = 0.39) or oral cavity (50% vs. 57%/58%; p = 0.94). CONCLUSION:p16-positivity confers a DFS benefit in larynx/hypopharynx/nasal cavity, but not in paranasal sinus or oral SCC.

Clinical characteristics of the study population.

MEAP exhibits superior multikinase inhibition compared to reversine

MEAP induces rapid STAT3 dephosphorylation in a NEDD9-dependent manner. A, Western blot illustrating the rapid kinetics at which MEAP selectively induces STAT3 dephosphorylation in THJ-16T ALDH+ cells. By comparison, MEAP inhibited Aurora-A nonselectively between ALDH+ and ALDH− cells. B, Immunoprecipitation experiment using anti-NEDD9. THJ16T ALDH+ cells were treated with 100 nmol/L MEAP treatment for the indicated duration and then harvested. C, Immunofluorescence images illustrating the impact of MEAP on pSTAT3705 and NEDD9 cellular distribution. D, Representative panel and quantification of NEDD9/pSTAT3705 colocalization in ALDH+ cells treated with DMSO or MEAP. E, Immunofluorescent images of ALDH−, ALDH+, and ALDH+ shNEDD9 cells stained for NEDD9 and pSTAT3705. F, Immunoblots illustrating MEAP’s ability to inhibit pSTAT3-705 in THJ-16T is dependent on NEDD9 expression. ALDH+ and ALDH− sorted cells were NEDD9-depleted or NEDD9-overexpressed and then treated with 100 nmol/L MEAP over multiple time points. (LZRS = LZRS_IRESGFP control, LZRS_NEDD9 = full-length NEDD9 cloned into LZRS-IRESGFP expression vector). G, Representative image of MEAP’s impact on colony formation in THJ-16T ALDH+ and ALDH− cells with stable NEDD9 depletion or NEDD9 overexpression, respectively (LZRS = LZRS_IRESGFP control, LZRS_NEDD9 = full-length NEDD9 cloned into LZRS-IRESGFP expression vector). Cells were seeded at 1,000 cells per well and grown for 5 days (n = 3). Scale bars, 10 μm.

OBJECTIVES:To report oncologic outcomes after metastasis-directed treatment (MDT) for metachronous thoracic metastases following definitive (chemo-)radiotherapy in p16-positive head and neck squamous cell carcinoma (HNSCC). METHODS:We reviewed all p16-positive HNSCC patients who developed metachronous thoracic metastases after definitive (chemo)radiotherapy (2003-2021). Metastatic disease was further classified as oligometastatic (1-5 lesions) versus polymetastatic (>5 lesions). Overall survival (OS) was compared between those receiving metastasis-directed therapy (MDT), either surgery or radiotherapy, and those managed without MDT. We transposed the TNM8 non-small-cell-lung-cancer staging algorithm to assess differences in outcome by MDT within the "lung-type stage I-III" subset. RESULTS:Among 1908 consecutively treated patients, 170 (9%) developed thoracic metastases, 98 (58%) of which were thorax-only. Of those, 34 (35%) had oligometastatic disease ("lung-type stage I-III"), of whom 24 received MDT: 18 surgery-only, 1 surgery + radiation, 4 radiation-only, and 1 chemoradiotherapy. Median follow-up was 2.0 years. Two-year OS was 88% with MDT versus 23% without (p < 0.001). On multivariable analysis, MDT was the only independent predictor of improved OS (adjusted hazard ratio 0.19, 95% CI 0.10-0.35, p < 0.001) adjusted for age, sex, disease site, and TNM8 stage group. Within the "lung-type stage I-III" subset, those who received MDT (n = 23/34) had higher 2-year OS versus those without MDT (87% versus 45%, p = 0.024). CONCLUSIONS:Patients with p16-positive HNSCC and metachronous thoracic oligometastatic disease who received MDT exhibit longer OS compared to those with polymetastatic disease or those without MDT. Future trials are warranted to validate the findings and to explore the optimal treatment approach for this population.

INTRODUCTION:Major salivary gland carcinoma (SGC) comprises diverse histologies with multiple interrelated risk factors, making it difficult to determine which patients truly benefit from post-operative radiotherapy (PORT). We developed a nomogram to predict the risk of locoregional failure (LRF) and model postoperative risk estimates under different PORT scenarios. MATERIAL AND METHODS:Major SGC patients treated with curative-intent surgery between 2000 and 2021 across five tertiary cancer centers were identified. Prognostic factors (p < 0.05) from multivariable analysis and clinicopathologically relevant characteristics were utilized to construct a nomogram estimating five-year LRF risk. Subsequently, we modeled the five-year LRF and overall survival (OS) risks for each nomogram point-value, with and without PORT. RESULTS:A total of 1175 patients were included in the analysis. The median follow-up was 5.3 years. The nomogram for prediction of five-year LRF risk comprised six statistically significant negative prognostic factors (lymphovascular invasion [100 points], WHO high-risk pathology [84], involved resection margins [61], parotid primary tumor [69], pathologic T3-4 category [44], and the non-utilization of PORT [57]), and two relevant factors (pathologic nodal involvement [34] and perineural invasion [7]). The corrected C-index was 0.77. We estimated the values of five-year LRF and OS depending on the PORT. For example, a patient with 271 points had a five-year LRF risk of 20% without PORT versus 11% with PORT, and a five-year OS of 65% versus 79%, respectively. DISCUSSION:This nomogram provides individualized estimates of LRF after surgery for major SGC and offers model-based risk estimates under different PORT scenarios. It may serve as a practical tool to guide personalized adjuvant treatment decisions.

BRAF V600 inhibitors are clinically approved for the treatment of BRAFV600-mutant melanoma in combination with a MEK inhibitor, but are ineffective in other melanoma subtypes. Moreover, pan-RAF inhibitors, such as belvarafenib, when combined with MEK inhibitors (cobimetinib), have promising but limited efficacy in non-BRAF-mutant melanomas. Here, we report that the mTOR inhibitor sapanisertib improves the efficacy of combined belvarafenib and cobimetinib therapy in NRAS, NF1, and KIT-mutant melanomas. Mechanistically, sapanisertib combined with belvarafenib and cobimetinib suppressed ATF4 expression and its target gene MTHFD2 while inducing DNA damage, revealing a previously underappreciated role of the ATF4-MTHFD2 axis in DNA damage repair and drug response. Human and murine models resistant to combined belvarafenib and cobimetinib exhibited elevated levels of ATF4 and MTHFD2 and were sensitive to sapanisertib. This study provides promising treatment opportunities for patients with non-BRAF-mutant melanomas, or those who relapse following belvarafenib and cobimetinib combination therapy.

MEAP eliminates ALDH+ ATC cells, increases PCM levels, induces spindle multipolarity, and reduces CIN in vivo. A–E, Orthotopic model; 5 × 105 8505c cells were injected into the right thyroid of NOD-SCID mice on day 0; mice were given 3 dosages of treated DMSO (vehicle), Taxol (5 mg/kg), or MEAP (30 mg/kg; n = 3–5) on day 15, 17, and 20 (i.p.); mice were sacrificed, and necropsy was performed on day 30. ALDH1A3 appeared as localized clusters in DMSO/Taxol-treated tumors which are absent in MEAP-treated tumors. A, Brightfield images IHC staining of ALDH1A3 staining in primary thyroid tumor. B, IHC staining of γ-tubulin in 8505c xenografts treated with DMSO, Taxol (5 mg/kg), or MEAP (30 mg/kg). C, Symmetry quantification, 10 centrosome pairs from each mouse was assessed. Symmetry is measured by comparing the relative intensity of γ-tubulin staining between centrosome pairs (intensity of weak/strong). To ensure cell-cycle parity, only centrosomes of equal distance are measured. D, Spindle multipolarity quantification. For spindle multipolarity, at least 50 mitotic cells from each mouse were evaluated (n = 4). E, Left, representative images representing chromosomal missegregation in 8505c xenografts, observed by hematoxylin and eosin (H&E) staining. Right, missegregated chromosome quantification. Based on H&E images (10 random field per mice, n = 4 for each group), quantification was done for the rate of mitotic cells exhibiting chromosome missegregation (lagging chromosomes or chromosome bridges). At least 50 mitotic cells from each mouse were evaluated (n = 4). F–H, Subcutaneous model; 1 × 106 8505c cells were implanted subcutaneously into NOD-SCID mice. Treatment started once tumor reached 40–80 mm3, and animals were treated three times a week (i.p.) with DMSO, Taxol (5 mg/kg), or MEAP (30 mg/kg; n = 8). F, Tumor size change curve of subcutaneous growth. G, Representative images of subcutaneous tumors were taken after necropsy. H, Initial and final body weights of animals from subcutaneous model. Scale bars, 10 μm. Data representation (mean ± SD) *, P < 0.05; **, P < 0.01 by one-way ANOVA with Tukey post hoc test.

Identification of MEAP as an inducer of centrosome-mediated MT nucleation in ATC cells with poorly nucleated supernumerary centrosomes. A, Experimental scheme of the MT regrowth assay. B, Chemical structure of MEAP. C, Quantification of relative centrosome MT nucleation activity in compound-treated THJ-16T cells based on α-tubulin staining intensity. At least 25 centrosomes were scored per experiment, n = 3 biological replicates. D, Representative images of MT regrowth in DMSO- or MEAP-treated THJ-16T cells with supernumerary centrosomes. Note that DMSO-treated cells exhibit supernumerary centrosomes with poor MT nucleation capacity, whereas MEAP-treated cells show robust MT nucleation at all supernumerary centrosomes. Scale bars, 10 μm. Data representation (mean ± SD). ***, P < 0.001; *****, P < 0.00001 by one-way ANOVA with Tukey post hoc test (Global P < 0.001). MEAP showed significantly higher centrosome MT nucleation activity than reversine (P < 0.01) and all other compounds tested.