Chuan-Xiang Zhou

Salivary adenoid cystic carcinoma (SACC) is a malignant salivary gland neoplasm characterized by aggressive local invasion and a marked propensity for metastasis. However, the role of MEOX1 in SACC progression remains poorly defined. In this study, we examined the effects of MEOX1 overexpression on the malignant behavior of SACC cells in vitro and in vivo. Human SACC-83 and SACC-LM cells were transduced with lentiviral vectors encoding MEOX1 or an empty vector control, and cell proliferation, migration, invasion, and cell cycle distribution were assessed using CCK-8, wound healing, Transwell, and flow cytometric assays, respectively. RNA sequencing was performed to characterize transcriptional changes associated with MEOX1 overexpression. In vivo, tumor growth was evaluated in BALB/c nude mice bearing subcutaneous xenografts, and pulmonary metastatic colonization was assessed using a tail vein injection model. MEOX1 overexpression reduced the proliferation, migration, and invasion of SACC cells in vitro and increased the G2/M phase fraction. In xenograft models, MEOX1-overexpressing cells formed smaller tumors and showed lower Ki67 staining than control cells. In the experimental lung metastasis model, mice injected with MEOX1-overexpressing cells developed fewer pulmonary metastatic nodules. RNA-seq identified 588 differentially expressed genes associated with MEOX1 overexpression, with enrichment in pathways including cytokine–cytokine receptor interaction, Toll-like receptor signaling, and G protein-coupled receptor signaling. Together, these findings indicate that enforced MEOX1 expression is associated with reduced malignant phenotypes in SACC models and with transcriptomic alterations in pathways related to immune response, G protein-coupled receptor signaling, and DNA damage response.

Adenoid cystic carcinoma (ACC) is a rare malignancy with 3 components (tubular, cribriform, and solid), and these components often coexist in the same patient. In the past few decades, great strides have been made in the pathological classification of ACC. However, due to the complexity of the pathological morphology, establishing a unified grading/assessment standard for clinical implementation process remains challenging. This study aimed to develop improved prognostic biomarkers to assist clinical pathological assessment at the molecular level. We used laser capture microdissection to isolate tumor microsamples with specific pathological features (solid, cribriform, and tubular component) and paracancerous normal glands from 100 patients, then performed DNA sequencing ( n = 955) and RNA sequencing ( n = 723) to obtain the genome and transcriptome profile, including copy number variation (CNV) type, gene fusion, and transcriptional expression. We found that CNV and gene fusion patterns differed among the 3 pathological subtypes and correlated with clinical outcome. We also found a mutually exclusive relationship between MYBL1::NFIB fusion and 6q-loss, and they displayed distinct different transcriptional profiles. We discovered that molecular markers 6q-loss and 14q-loss were strongly associated with poor prognosis in ACC. Patients with 6q-loss or 14q-loss had a higher risk of tumor metastasis and recurrence, whereas MYB::NFIB and MYBL1::NFIB fusions showed no adverse prognostic impact. Based on these findings, we propose a new CNV-based molecular classification that stratifies patients into the subgroups of 6q-loss, 14q-loss, and others. The molecular classification method increased the prognostic accuracy from 74.49% (the pathological method) to now 82.65%, especially in cribriform patients (increased from 64% to 80%). Fluorescence in situ hybridization–based detection of 6q-loss and 14q-loss by probe ESR1-6q25 and FOS-14q24 provided a rapid and clinically feasible validation in ACC prognostic assessment. Further multicohort and multifactor analysis demonstrated the robustness and independence of the prognostic value in overall survival and metastasis-free survival of the molecular classification method.

Acinic cell carcinoma of the salivary gland (AciCC) is a low-grade carcinoma characterized by the overexpression of the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3). AciCC has been the subject of a few molecular research projects. This study delves into AciCC’s molecular landscape to identify additional alterations and explore their clinical implications. RNA sequencing and immunohistochemical staining for markers NR4A3/NR4A2, DOG-1, S100, and mammaglobin were utilized on 41 AciCCs and 11 secretory carcinoma (SC) samples. NR4A3 was evident in 35 AciCCs, while the residual 6 were NR4A3-negative and NR4A2-positive; SC samples were consistently NR4A3-negative. A novel fusion, PON3 exon 1–LCN1 exon 5, was detected in 9/41 (21.9%) AciCCs, exhibiting a classical histologic pattern with serous cell components growing in solid sheets alongside the intercalated duct-like component. Clinical follow-up of 39 patients over a median of 59 months revealed diverse prognostic outcomes: 34 patients exhibited no disease evidence, whereas the remaining 5 experienced poorer prognosis, involving local recurrence, lymph node, and distant metastasis, and disease-associated death, 4 of which harbored the PON3::LCN1 fusion. In addition, the HTN3::MSANTD3 fusion was recurrently identified in 7/41 AciCC cases. SC patients lacked both fusions. Immunohistochemistry uncovered differential expression of DOG-1, S100, and mammaglobin across samples, providing nuanced insights into their roles in AciCC. This study accentuates PON3::LCN1 and HTN3::MSANTD3 fusions as recurrent molecular events in AciCC, offering potential diagnostic and prognostic utility and propelling further research into targeted therapeutic strategies.

Background: Engrailed homeobox 1 (EN1) is a candidate oncogene that is epigenetically modified in salivary adenoid cystic carcinoma (SACC). We investigated the expression of EN1 in SACC tissues and cells, EN1 promoter methylation, and the role of EN1 in tumour progression in SACC. Methods: Thirty-five SACC samples were screened for key transcription factors that affect tumour progression. In vitro and in vivo assays were performed to determine the viability, tumorigenicity, and metastatic ability of SACC cells with modulated EN1 expression. Quantitative methylation-specific polymerase chain reaction analysis was performed on SACC samples. Results: EN1 was identified as a transcription factor that was highly overexpressed in SACC tissues, regardless of clinical stage and histology subtype, and its level of expression correlated with distant metastasis. EN1 promoted cell invasion and migration through epithelial-mesenchymal transition in vitro and enhanced SACC metastasis to the lung in vivo. RNA-seq combined with in vitro assays indicated that EN1 might play an oncogenic role in SACC through the PI3K-AKT pathway. EN1 mRNA levels were negatively correlated with promoter hypermethylation, and inhibition of DNA methylation by 5-aza-dC increased EN1 expression. Conclusions: The transcription factor EN1 is overexpressed in SACC under methylation regulation and plays a pivotal role in SACC progression through the PI3K-AKT pathway. These results suggest that EN1 may be a diagnostic biomarker and a potential therapeutic target for SACC.

The relationship between various patterns of mucin-producing salivary adenocarcinomas, including invasive salivary adenocarcinomas with mucinous differentiation, such as colloid and papillary carcinomas, remains unclear. Herein, we aimed to describe the clinicopathologic characteristics, immunophenotypes, molecular underpinnings, and clinical behavior of salivary mucinous adenocarcinomas (MA) to clarify their classification. We described a broad series of colloid and papillary patterns of MAs, indicating that papillary pattern presented papillary cystic proliferation of mucinous columnar cells as salivary intraductal papillary mucinous neoplasms with recurrent AKT1 E17K mutations, whereas colloid adenocarcinomas containing large mucinous pools or lakes around the malignant epithelial nests or islands harbored BRAF V600E mutations with worse prognosis. Typical morphologic structures, CK7(+), CK20(–), CDX2(–), p63(–), p40(–), MAML2 fluorescence in situ hybridization (–), AR(–), TTF-1(–), S100(–), mammaglobin(–), or S100/mammaglobin(+) with ETV6 fluorescence in situ hybridization (–) immunophenotype, and recurrent AKT1 E17K or BRAF V600E mutations may be defined. To our knowledge, this small series represents the first genetic study on a typical colloid pattern of MA, and our study with the spectrum documentation for MA in clinicopathologic characteristics, histologic and immunophenotypes, molecular features, and clinical behavior will allow for a better understanding of these rare but distinctive tumors.

Background: Familial gigantiform cementoma (FGC) is a rare tumor characterized by the early onset of multi‐quadrant fibro‐osseous lesions in the jaws, causing severe maxillofacial deformities. Its clinicopathological features overlap with those of other benign fibro‐osseous lesions. FGC eventually exhibits progressively rapid growth, but no suspected causative gene has been identified.Methods: In this study, three patients with FGC were recruited, and genomic DNA from the tumor tissue and peripheral blood was extracted for whole‐exome sequencing.Results: Results showed that all three patients harbored the heterozygous mutation c.1067G > A (p.Cys356Tyr) in the ANO5 gene. Furthermore, autosomal dominant mutations in ANO5 at this locus have been identified in patients with gnathodiaphyseal dysplasia (GDD) and are considered a potential causative agent, suggesting a genetic association between FGC and GDD. In addition, multifocal fibrous bone lesions with similar clinical presentations were detected, including five cases of florid cemento‐osseous dysplasia, five cases of polyostotic fibrous dysplasia, and eight cases of juvenile ossifying fibromas; however, none of them harbored mutations in the ANO5 gene.Conclusion: Our findings indicate that FGC may be an atypical variant of GDD, providing evidence for the feasibility of ANO5 gene testing as an auxiliary diagnostic method for complex cases with multiple quadrants.

[This corrects the article on p. 514 in vol. 5, PMID: 25973294.].

Background: Salivary gland tumors with papillary architecture and intestinal‐like mucinous cytologic features are rare. Their clinicopathologic and genetic features are not fully understood, and whether they represent one separate entity remains unclear.Methods: Six salivary adenocarcinomas with papillary architecture and intestinal‐like mucinous cytologic features were reported. Immunostaining was done for CK7, CK20, CDX2, SOX10, S100, MUC1, MUC2, and MUC5AC. Tumor DNA samples were extracted for Sanger sequencing. Previously reported morphology‐analogous cases were reviewed.Results: Six cases involved the palate (2), retromolar region (1), submandibular region (1), tongue (1), and mandible (1). Five cases were followed up, with one case of recurrence 1 year after surgery, one death from cerebral infarction 7 days after surgery, and three cases without signs of recurrence or metastasis over 5 years. All cases had abundant mucinous production and presented a typical immunophenotype common to salivary primaries, CK7 & MUC1 positive, CK20 & CDX2 negative. Sanger sequencing demonstrated recurrent AKT1 E17K mutations in four cases (4/6, 66.7%). A review of reported salivary intestinal‐like tumors revealed 3 out of 13 cases presented with papillary morphology and CDX2 negative. Some salivary papillary neoplasms with mucinous cytologic features termed as intraductal papillary neoplasms or mucinous adenocarcinomas were also reported with AKT1 E17K mutations.Conclusion: We describe 6 cases of salivary gland papillary adenocarcinoma with intestinal‐like mucinous cytologic features, which are different from conventional intestinal‐type adenocarcinoma, presenting a consistent immunophenotype of CK7 & MUC1 positive, CK20 & CDX2 negative and exhibiting recurrent AKT1 E17K mutations.

Objective: We aimed to investigate Notch pathway dysregulation in solid adenoid cystic carcinoma (AdCC) and to define the association of Notch activation with cell differentiation and prognosis in AdCCs.Materials and Methods: Notch1 mutations were detected from 125 AdCCs (62 cribriform‐tubular; 63 solid). RNA‐seq was performed in 16 AdCCs (6 Notch‐mutant; 10 wild type). Notch activation indicator NICD and myoepithelial marker p63 were detected using immunohistochemistry and double‐labelling immunofluorescence. The effect of exogenous NICD overexpression on p63 expression and cell proliferation was investigated using Western blotting and live‐cell imaging.Results: We identified 33 Notch1 activating mutations in 27 AdCCs including 26 solid and 1 cribriform‐tubular subtypes. Six tumours harboured more than one Notch1 mutation, and 18 Notch1 mutations were novel. Most (47/63, 74.6%) solid AdCCs showed NICD overexpression, whereas 61 of 62 (98.4%) cribriform‐tubular tumours were negative. NICD and p63 exhibited mutually exclusive expression, and exogenous NICD overexpression promoted cell proliferation and decreased p63 expression. NICD overexpression and Notch mutations were poor indicators for overall survival and metastasis, especially bone metastasis.Conclusions: Dysregulated Notch signalling plays a critical role in AdCC severity. Notch activation may contribute to loss of myoepithelial differentiation as well as high proliferation and metastasis rates in solid AdCC.

回顾性分析唾液腺腺样囊性癌颈部淋巴结转移的发病率及其临床病理特征。 回顾性分析2001年1月至2019年1月在北京大学口腔医院诊治并经病理确诊为腺样囊性癌的798例患者的临床病理资料,对82例发生颈部淋巴结转移的患者进行详细的临床病理及统计学分析。 纳入研究的腺样囊性癌患者798例,腭部、舌下口底区域和腮腺区是腺样囊性癌常见的发病部位。经病理学检查证实发生颈部淋巴结转移者82例,总体转移率约10.3%(82/798),其中原发于颌下腺、舌下口底区及舌部的颈部淋巴结转移率较高,分别为20.8%、16.1%、15.1%,而腭部、腮腺原发病例颈部淋巴结转移率较低,分别为6.1%、3.4%。颈Ⅰ、Ⅱ区为常见转移区域,极少累及Ⅳ、Ⅴ区淋巴结。主要转移模式是由癌细胞沿经典的淋巴管/血管通路转移(70.7%),部分颌下及舌下病例不除外直接侵犯淋巴结。82例伴淋巴结转移的患者5年和10年总生存率分别为77.4% 和 20.6%。淋巴结转移阳性组患者总生存率和无瘤生存率均显著低于转移阴性组,且组织学呈现实性型的腺样囊性癌颈部淋巴结转移率显著高于筛孔型和管状型肿瘤。 舌下、口底及颌下腺为唾液腺腺样囊性癌颈部淋巴转移常见原发部位,实性型组织学结构与转移率显著正相关,在临床诊治及随访中需要更多关注。