High FOXA1 levels induce ER transcriptional reprogramming, a pro-metastatic secretome, and metastasis in endocrine-resistant breast cancer.

Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, the key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) enriched in ER+ endocrine-resistant (EndoR) cells. We identify Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, as a strong predictor for poor outcomes of ER+ BC. It is elevated in ER+ metastases vs. primary tumors, irrespective of ESR1 mutations. Genomic ER binding near Secretome14 genes is also increased in mutant ER-expressing or mitogen-treated ER+ BC cells and in ER+ metastatic vs. primary tumors, suggesting a convergent pathway including high growth factor receptor signaling in activating pro-metastatic secretome genes. Our findings uncover H-FOXA1-induced ER reprogramming that drives EndoR and metastasis partly via an H-FOXA1/ER-dependent secretome.

[Minimally invasive osteotomy with absorbable screws in treating hallux valgus deformity].

OBJECTIVE: To evaluate clnical effect of minimally invasive osteotomy with absorbable screws in treating hallux valgus deformity. METHODS: Clnical data of 31 patients with hallux valgus deformity were retrospective analyzed from January 2019 to December 2020, and divided into absorbable screws group (17 patients) and titanium cannulated screw group (14 patients). In absorbable screws group, there were 1 male and 16 females aged from 32 to 72 years old with an average of (54.53±12.12) years old;6 patients on the left side, 5 on bilateral and 6 on the right side;1 patient was mild, 11 moderate and 5 severe;treated with minimally invasive osteotomy and fixation of absorbable screws. In titanium cannulated screw group, there were 2 males and 12 females aged from 18 to 71 years old with an average of (47.57±15.68) years old;4 patients on the left side, 4 on bilateral and 6 on the right side;1 patient was mild, 9 moderate and 4severe;treated with minimally invasive osteotomy and fixation of titanium cannulated screw. Complications between two groups were observed, changes of hallux valgus angle (HVA)and intermetatarsal angle (IMA)were detected and compared before and after operation at 12 months, American Orthopedic Foot and Ankle Society(AOFAS) and visual analogue scale(VAS) before and after operation at 12 months were also compared. RESULTS: All 31 patients were followed up from 13 to 20 months with an average of (16.61±2.47) months. Patients in absorbable screws group were followed up from 14 to 20 months with an average of (16.88±2.80) months, while patients in titanium cannulated screw group were followed up from 13 to 19 months with an average of (16.29±2.05) months;there was no difference between two groups(P>0.05). One patient in absorbable screws group occurred numbness around incision, 3 patients in titanium cannulated screw group occurred complications, including numbness around incision in 1 patient, skin irritation due to internal fixation in 1 patient, and recurrence in 1 case;there was no statistic difference between two groups (χ2=1.651, P=0.199). There were no statistic difference in HVA and IMA between two groups before and after operation at 12 months(P>0.05). There were no statistic difference between two groups in AOFAS and VAS before and after operation at 12 months(P>0.05). CONCLUSION: Compare with mainstream fixation with titanium hollow screw after minmally invasive osteotomy, fixation with absorbable screw could achieve comparable clinical outcome on the basis of images and function evaluation.

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor-positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1-estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1-ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. SIGNIFICANCE: A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.

NPY1R exerts inhibitory action on estradiol-stimulated growth and predicts endocrine sensitivity and better survival in ER-positive breast cancer.

G Protein-Coupled Receptors (GPCRs) represent the largest superfamily of cell-surface proteins. However, the expression and function of majority of GPCRs remain unexplored in breast cancer (BC). We interrogated the expression and phosphorylation status of 398 non-sensory GPCRs using the landmark BC proteogenomics and phosphoproteomic dataset from The Cancer Genome Atlas. Neuropeptide Y Receptor Y1 (NPY1R) gene and protein expression were significantly higher in Luminal A tumors versus other BC subtypes. The trend of NPY1R gene, protein, and phosphosite (NPY1R-S368s) expression was decreasing in the order of Luminal A, Luminal B, Basal, and human epidermal growth factor receptor 2 (HER2) subtypes. NPY1R gene expression increased in response to estrogen and reduced with endocrine therapy in estrogen receptor-positive (ER+) BC cells and xenograft models. Conversely, NPY1R expression decreased in ER+ BC cells resistant to endocrine therapies (estrogen deprivation, tamoxifen, and fulvestrant) in vitro and in vivo. NPY treatment reduced estradiol-stimulated cell growth, which was reversed by NPY1R antagonist (BIBP-3226) in ER+ BC cells. Higher NPY1R gene expression predicted better relapse-free survival and overall survival in ER+ BC. Our study demonstrates that NPY1R mediates the inhibitory action of NPY on estradiol-stimulated growth of ER+ BC cells, and its expression serves as a biomarker to predict endocrine sensitivity and survival in ER+ BC patients.

Temperature impacts fate of antibiotic resistance genes during vermicomposting of domestic excess activated sludge.

Effect of temperature on antibiotic resistance genes (ARGs) during vermicomposting of domestic excess sludge remains poorly understood. Vermicomposting experiment with excess sludge was conducted at three different temperatures (15 °C, 20 °C, and 25 °C) to investigate the fate of ARGs, bacterial community and their relationship in the process. The vermicomposting at 25 °C did not significantly attenuate the targeted ARGs relative to that at 15 °C and 20 °C. The dynamics of qnrA, qnrS, and tetM genes during vermicomposting at 15 °C and 20 °C followed the first-order kinetic model. Temperature remarkably impacted bacterial diversity of the final products with the lowest Shannon index at 25 °C. The presence of the genus (Aeromonas and Chitinophagaceae) at 25 °C may contribute to the rebound of the genes (qnrA, qnrS and tetM). The study indicates that 20 °C is a suitable vermicomposting temperature to simultaneously reach the highest removal efficiency of the ARGs and the good biostability of the final product.

Parental exposure 3-methylcholanthrene disturbed the enterohepatic circulation in F1 generation of mice.

3-methylcholanthrene (3 MC) is an environmental compound belonging to the PAHs and is reportedly thought to be a risk factor for the prevalence of hepatic function disorder. Here, a dose of 0.5 mg/kg of 3 MC was given to 4-week-old male and female mice (F0) in their diet for 6 weeks. After exposure, then the mice were mated between different groups. The first filial (F1) generation offspring of exposed or unexposed parental mice were sacrificed at the age of 5 weeks (F1-5 W), and the potential effects on the F0 and F1 offspring were evaluated. The results showed that the total bile acids (TBAs) in the serum and feces in F0 females and female F1-5 W individuals born from female mice exposed to 3 MC decreased, while the TBAs in the liver increased. The transcriptional levels of major genes participating in synthesis, regulation, transportation and apical uptake was also altered correspondingly. In addition, the transcription of some genes related to inflammation was enhanced in these mice. Further investigation revealed that in addition to distinct changes in the mucus secretion, tight junction proteins and ion transport were induced, and antimicrobial peptides were also disrupted in the intestine of F0 mice and F1-5 W female offspring of maternal mice exposed to 3 MC. Our results suggested that exposure to 3 MC, but not male exposure, had the potential to interfere with BAs metabolism, affecting gut barrier function. Females were more seriously affected than males.

Interferon Signaling in Estrogen Receptor-positive Breast Cancer: A Revitalized Topic.

Cancer immunology is the most rapidly expanding field in cancer research, with the importance of immunity in cancer pathogenesis now well accepted including in the endocrine-related cancers. The immune system plays an essential role in the development of ductal and luminal epithelial differentiation in the mammary gland. Originally identified as evolutionarily conserved antipathogen cytokines, interferons (IFNs) have shown important immune-modulatory and antineoplastic properties when administered to patients with various types of cancer, including breast cancer. Recent studies have drawn attention to the role of tumor- and stromal-infiltrating lymphocytes in dictating therapy response and outcome of breast cancer patients, which, however, is highly dependent on the breast cancer subtype. The emerging role of tumor cell-inherent IFN signaling in the subtype-defined tumor microenvironment could influence therapy response with protumor activities in breast cancer. Here we review evidence with new insights into tumor cell-intrinsic and tumor microenvironment-derived IFN signaling, and the crosstalk of IFN signaling with key signaling pathways in estrogen receptor-positive (ER+) breast cancer. We also discuss clinical implications and opportunities exploiting IFN signaling to treat advanced ER+ breast cancer.

Activation of the IFN Signaling Pathway is Associated with Resistance to CDK4/6 Inhibitors and Immune Checkpoint Activation in ER-Positive Breast Cancer.

PURPOSE: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor-positive (ER+)/HER2- breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. EXPERIMENTAL DESIGN: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation-resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. RESULTS: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an "IFN-related palbociclib-resistance Signature" (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. CONCLUSIONS: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Changes in physicochemical properties and microfauna community during vermicomposting of municipal sludge under different moisture conditions.

The present study aimed to explore the effect of a range of moisture content levels, including 65%, 72%, and 78%, on physicochemical properties and microfauna communities during vermicomposting of municipal sludge. As a result, death of perishable microfauna together with the degradation of organic matter was the dominant response in all groups in the early period of vermicomposting, while the effects of moisture content levels on various physiochemical parameters did not appear until the mid-later period. After the treatment with 78% moisture content, the content of mineral nitrogen was 1.186 g/kg in the sludge, with a 9.36 × 103 ind./g of microfauna quantity and 663.01 g of earthworm biomass. The values of these three measurements in 78% group were significantly higher than other two groups (p < 0.05), indicating that the effects of 78% moisture content were more pronounced for promoting nitrogen mineralization as well as microfauna and earthworms growth during vermicomposting. Specifically, testate amoebae were strongly associated with nitrification process, while nematodes were related to ammonification and phosphorus mineralization, of which testate amoebae had great potential of being bioindicators during vermicomposting of municipal sludge.

Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance.

Acquired therapy resistance is a major problem for anticancer treatment, yet the underlying molecular mechanisms remain unclear. Using an established breast cancer cellular model, we show that endocrine resistance is associated with enhanced phenotypic plasticity, indicated by a general downregulation of luminal/epithelial differentiation markers and upregulation of basal/mesenchymal invasive markers. Consistently, similar gene expression changes are found in clinical breast tumours and patient-derived xenograft samples that are resistant to endocrine therapies. Mechanistically, the differential interactions between oestrogen receptor α and other oncogenic transcription factors, exemplified by GATA3 and AP1, drive global enhancer gain/loss reprogramming, profoundly altering breast cancer transcriptional programs. Our functional studies in multiple culture and xenograft models reveal a coordinated role of GATA3 and AP1 in re-organizing enhancer landscapes and regulating cancer phenotypes. Collectively, our study suggests that differential high-order assemblies of transcription factors on enhancers trigger genome-wide enhancer reprogramming, resulting in transcriptional transitions that promote tumour phenotypic plasticity and therapy resistance.

Author Correction: Temporal dynamic reorganization of 3D chromatin architecture in hormone-induced breast cancer and endocrine resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery, Structure-Activity Relationship, and Biological Activity of Histone-Competitive Inhibitors of Histone Acetyltransferases P300/CBP.

Histone acetyltransferase (HAT) p300 and its paralog CBP acetylate histone lysine side chains and play critical roles in regulating gene transcription. The HAT domain of p300/CBP is a potential drug target for cancer. Through compound screening and medicinal chemistry, novel inhibitors of p300/CBP HAT with their IC50 values as low as 620 nM were discovered. The most potent inhibitor is competitive against histone substrates and exhibits a high selectivity for p300/CBP. It inhibited cellular acetylation and had strong activity with EC50 of 1-3 µM against proliferation of several tumor cell lines. Gene expression profiling in estrogen receptor (ER)-positive breast cancer MCF-7 cells showed that inhibitor treatment recapitulated siRNA-mediated p300 knockdown, inhibited ER-mediated gene transcription, and suppressed expression of numerous cancer-related gene signatures. These results demonstrate that the inhibitor is not only a useful probe for biological studies of p300/CBP HAT but also a pharmacological lead for further drug development targeting cancer.

Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity.

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.

In vivo longitudinal imaging of RNA interference-induced endocrine therapy resistance in breast cancer.

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor-positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence-labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)-regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox-induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor-bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen-treated control group. Longitudinal imaging was performed by a home-built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper-measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e-7) correlated with the tumor size progression for the mice with shRNA-induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.

FOXA1 upregulation promotes enhancer and transcriptional reprogramming in endocrine-resistant breast cancer.

Forkhead box A1 (FOXA1) is a pioneer factor that facilitates chromatin binding and function of lineage-specific and oncogenic transcription factors. Hyperactive FOXA1 signaling due to gene amplification or overexpression has been reported in estrogen receptor-positive (ER+) endocrine-resistant metastatic breast cancer. However, the molecular mechanisms by which FOXA1 up-regulation promotes these processes and the key downstream targets of the FOXA1 oncogenic network remain elusive. Here, we demonstrate that FOXA1 overexpression in ER+ breast cancer cells drives genome-wide enhancer reprogramming to activate prometastatic transcriptional programs. Up-regulated FOXA1 employs superenhancers (SEs) to synchronize transcriptional reprogramming in endocrine-resistant breast cancer cells, reflecting an early embryonic development process. We identify the hypoxia-inducible transcription factor hypoxia-inducible factor-2α (HIF-2α) as the top high FOXA1-induced SE target, mediating the impact of high FOXA1 in activating prometastatic gene sets and pathways associated with poor clinical outcome. Using clinical ER+/HER2- metastatic breast cancer datasets, we show that the aberrant FOXA1/HIF-2α transcriptional axis is largely nonconcurrent with the ESR1 mutations, suggesting different mechanisms of endocrine resistance and treatment strategies. We further demonstrate the selective efficacy of an HIF-2α antagonist, currently in clinical trials for advanced kidney cancer and recurrent glioblastoma, in reducing the clonogenicity, migration, and invasion of endocrine-resistant breast cancer cells expressing high FOXA1. Our study has uncovered high FOXA1-induced enhancer reprogramming and HIF-2α-dependent transcriptional programs as vulnerable targets for treating endocrine-resistant and metastatic breast cancer.

Targeting the Mevalonate Pathway to Overcome Acquired Anti-HER2 Treatment Resistance in Breast Cancer.

Despite effective strategies, resistance in HER2+ breast cancer remains a challenge. While the mevalonate pathway (MVA) is suggested to promote cell growth and survival, including in HER2+ models, its potential role in resistance to HER2-targeted therapy is unknown. Parental HER2+ breast cancer cells and their lapatinib-resistant and lapatinib + trastuzumab-resistant derivatives were used for this study. MVA activity was found to be increased in lapatinib-resistant and lapatinib + trastuzumab-resistant cells. Specific blockade of this pathway with lipophilic but not hydrophilic statins and with the N-bisphosphonate zoledronic acid led to apoptosis and substantial growth inhibition of R cells. Inhibition was rescued by mevalonate or the intermediate metabolites farnesyl pyrophosphate or geranylgeranyl pyrophosphate, but not cholesterol. Activated Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and mTORC1 signaling, and their downstream target gene product Survivin, were inhibited by MVA blockade, especially in the lapatinib-resistant/lapatinib + trastuzumab-resistant models. Overexpression of constitutively active YAP rescued Survivin and phosphorylated-S6 levels, despite blockade of the MVA. These results suggest that the MVA provides alternative signaling leading to cell survival and resistance by activating YAP/TAZ-mTORC1-Survivin signaling when HER2 is blocked, suggesting novel therapeutic targets. MVA inhibitors including lipophilic statins and N-bisphosphonates may circumvent resistance to anti-HER2 therapy warranting further clinical investigation. IMPLICATIONS: The MVA was found to constitute an escape mechanism of survival and growth in HER2+ breast cancer models resistant to anti-HER2 therapies. MVA inhibitors such as simvastatin and zoledronic acid are potential therapeutic agents to resensitize the tumors that depend on the MVA to progress on anti-HER2 therapies.

Gut digestion of earthworms significantly attenuates cell-free and -associated antibiotic resistance genes in excess activated sludge by affecting bacterial profiles.

Vermicomposting can significantly attenuate antibiotic resistance genes (ARGs) in the excess activated sludge (EAS). However, the effect of earthworms, especially the effect of gut digestion as a critical step in the vermicomposting process, remains unclarified. The purpose of this study was to investigate the response of ARGs (cell-free and -associated) in EAS to gut digestion of earthworms and to clarify the possible mechanism from the viewpoint of bacterial community through quantitative polymer chain reaction (q-PCR) and high throughput sequencing. Compared to the initial sludge, the earthworm casts were observed to have significantly lower absolute abundances of ARGs, especially qnrS, tetM, and tetX with the removal exceeding 90%. Cell-free and -associated ARGs (except sul1 and tetG) had equivalent contributions to the attenuation of each ARG. Remarkable reductions of bacterial number and alpha diversity (chao1 and Shannon) were detected in the casts. Spearman correlation analysis between the targeted genes and bacterial community indicates that twelve different phyla mainly including Acidobacteria, Euryarchaeota, Deinococcus-Thermus, Chlorobi, Firmicutes, Fibrobacteres, and Proteobacteria are the potential ARGs hosts, suggesting that the fate and behaviour of these hosts during gut digestion of EAS by earthworms substantially determined the dynamics of the ARGs. These findings increase our understanding of earthworm gut digestion as an important process for the attenuation of ARGs in EAS, and contribute towards preventing their release into the total environment.

Temporal dynamic reorganization of 3D chromatin architecture in hormone-induced breast cancer and endocrine resistance.

Recent studies have demonstrated that chromatin architecture is linked to the progression of cancers. However, the roles of 3D structure and its dynamics in hormone-dependent breast cancer and endocrine resistance are largely unknown. Here we report the dynamics of 3D chromatin structure across a time course of estradiol (E2) stimulation in human estrogen receptor α (ERα)-positive breast cancer cells. We identified subsets of temporally highly dynamic compartments predominantly associated with active open chromatin and found that these highly dynamic compartments showed higher alteration in tamoxifen-resistant breast cancer cells. Remarkably, these compartments are characterized by active chromatin states, and enhanced ERα binding but decreased transcription factor CCCTC-binding factor (CTCF) binding. We finally identified a set of ERα-bound promoter-enhancer looping genes enclosed within altered domains that are enriched with cancer invasion, aggressiveness or metabolism signaling pathways. This large-scale analysis expands our understanding of high-order temporal chromatin reorganization underlying hormone-dependent breast cancer.

Implications of Classification of Os Trigonum: A Study Based on Computed Tomography Three-Dimensional Imaging.

BACKGROUND The os trigonum is an accessory bone that is not fully fused with the talus during secondary ossification, and is one of the risk factors of posterior malleolus impact syndrome. The purpose of this study was to classify the os trigonum and to guide the diagnosis and treatment of related clinical diseases. MATERIAL AND METHODS Ankle computed tomography (CT) scans of 586 Chinese patients between October 2014 and October 2018 were reviewed. CT images of 1011 ankle joints were reconstructed to evaluate the classification of the os trigonum and the measurement of anatomical parameters. RESULTS The incidences of os trigonum in 3 groups were determined as type I (1.9%), type II (10.5%), and type III (14.7%). The macro-axis of type II (0.89±0.31) cm was significantly larger than with type I (0.65±0.24 cm) and type III (0.74±0.23 cm) (p<0.05).The minor axis of similar of type I (0.41±0.23 cm) was significantly shorter than that of type II (0.58±0.32 cm) and type III (0.55±0.16 cm) (p<0.05).The distance from os trigonum to calcaneal tubercle was significantly different than that of type I (1.33±0.52 cm), type II (1.67±0.55 cm), and type III (1.84±0.45 cm) (p<0.05). CONCLUSIONS This study showed that os trigonum has a high incidence. Type I was the least common, the volume of type II was larger, and type III was more common. The anatomical parameters of each type may improve treatment of related diseases and the further development of ankle arthroscopic surgery.