This study analyzed the effect of serum containing Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) herb pair on prostate cancer(PCa) cell proliferation, migration, apoptosis, and immune micro-environment based on the androgen receptor(AR)/forkhead box protein A1(FOXA1) signaling axis and tumor-associated macrophages(TAMs) polarization. SD rats were gavaged with SR, CR to obtain SR-CR-containing serum. Cell counting kit-8(CCK-8), colony formation, wound-healing, and 3D tumor sphere assays were conducted to detect the synergistic inhibitory effect of SR-CR-containing serum on the proliferation and migration of 22RV1 and C4-2B cells. Western blot was employed to examine AR and FOXA1 protein levels. A RAW264.7-C4-2B co-culture model combined with flow cytometry was used to evaluate the effect of SR-CR on macrophage M2 polarization(CD206) and cancer cell apoptosis(Annexin V-FITC/PI). Further, the RM-1 subcutaneous transplanted tumor model was established in C57BL/6J mice to observe the regulation of SR-CR combined medication on tumor volume, spleen index, and intratumoral M2-TAMs ratio. Compared with the single medication group, the results indicated the following changes after 10% SR + 10% CR containing serum intervention for 48 hours. Proliferation, colony formation, and migration ability were significantly lower in two PCa cells(P<0.01)(CI<1), exhibiting a strong synergistic effect. AR and FOXA1 expression was significantly down-regulated(P<0.05), and 3D tumor sphere volume was decreased(P<0.05). In the co-culture system, the M2-TAMs ratio was significantly reduced while cancer cell apoptosis was significantly increased(P<0.01). In vivo experiment demonstrated that, compared with the control group, the SR-CR synergy group displayed lower tumor weight and tumor volume, elevated spleen index, and fewer M2-TAMs within tumors(all P<0.05). In summary, the SR-CR herbal pair, through mutual reinforcement(Xiang-Xu), concurrently inhibits the AR/FOXA1 signaling axis and arrests M2-TAMs polarization, thereby enhancing anti-PCa efficacy.

The Hippo signaling pathway, as a highly conserved tumor suppressor pathway, is closely related to the occurrence and development of various malignant tumors due to the abnormal expression of its core components, mammalian STE20-like protein kinase 1/2(MST1/2), large tumor suppressor kinase 1/2(LATS1/2), and effector factor Yes-associated protein 1(YAP)/WW domain-containing transcription regulator 1(TAZ). This pathway plays a key regulatory role in pathological processes such as malignant proliferation, apoptosis, and drug resistance by regulating downstream target genes such as connective tissue growth factor(CTGF) and cysteine-rich angiogenic inducer 61(CYR61). Currently, it has been found through research that effective components of traditional Chinese medicine(TCM) and TCM formulas can exert anti-cancer effects by regulating the Hippo signaling pathway, but the specific targets and biochemical processes involved remain unknown. This article integrated the action targets and regulated biochemical processes of effective components of TCM targeting the Hippo signaling pathway in anti-tumor activities and discovered that flavonoids, terpenoids, glycosides, and other effective components of TCM and TCM formulas can regulate the Hippo signaling network, with the most significant mechanisms being the intervention of YAP/TAZ nuclear-cytoplasmic shuttling, restoration of the activity of Hippo core kinases, and blocking of crosstalk between pathways. Taking the Hippo pathway as the research object not only provides a new perspective for explaining the scientific connotation of TCM in anti-tumor activities but also lays a theoretical foundation for the development of new TCM drugs based on Hippo pathway-targeted therapy.

This study aimed to investigate whether Xihuang Pills(XHP) enhance the therapeutic efficacy of temozolomide(TMZ) against glioblastoma(GBM) U251 cells by inducing mitochondria-associated ferroptosis via regulation of Nrf2/HO-1/GPX4 signaling axis. The XHP-containing serum was prepared, and the effects of various concentrations of TMZ(0-200 μmol·L~(-1)) and XHP(0-20%) on U251 cell viability were assessed by CCK-8 assay. The optimal synergistic dose(200 μmol·L~(-1) TMZ + 10% XHP) was identified via the SynergyFinder platform for establishing a combined intervention model. Control, XHP, TMZ, and XHP + TMZ groups were designed. Cell proliferation was evaluated by EdU staining and colony formation assay, and cell migration and invasion were assessed by Transwell and wound healing assays. FerroOrange and DCFH-DA fluorescent probes were used to measure the intracellular levels of Fe~(2+) and ROS, respectively. The JC-1 fluorescent probe was used to assess mitochondrial membrane potential, while Mito-PeDPP fluorescent probe to examine mitochondrial lipid peroxidation. Mito-Tracker Green and MitoSOX~(TM) Red were used to examine mitochondrial morphology and oxidative stress. Transmission electron microscopy was adopted to observe the mitochondrial ultrastructure. GSH and MDA levels were measured by colorimetric assays. Western blot was employed to assess the protein levels of Nrf2, HO-1, xCT, and GPX4. The results showed that, XHP + TMZ suppressed cell viability, proliferation, migration, and invasion(P<0.01), increased Fe~(2+) accumulation, ROS generation, GSH depletion, and lipid peroxidation(P<0.001), reduced mitochondrial membrane potential, caused severe structural damage, and down-regulated the protein levels of Nrf2, HO-1, GPX4, and xCT, which indicated that the antioxidant ferroptosis-protective pathway was co-inhibited. In conclusion, XHP enhances TMZ-induced ferroptosis in GBM cells by suppressing the Nrf2/HO-1/GPX4 signaling axis. The findings provide new mechanism insights into the synergistic antitumor effect of traditional Chinese medicine in glioblastoma therapy and offer experimental evidence for the treatment of refractory brain tumors with combined strategies.

This article aims to investigate the inhibitory effect and molecular mechanism of sculponeatin A(STA) on triple negative breast cancer(TNBC). MDA-MB-436 and MDA-MB-468 were selected as cell models. The MTT assay, real-time cell analysis(RTCA), and colony formation assay were used to evaluate the effects of different concentrations of STA on the proliferation of TNBC cells. JC-1 staining and Annexin V-FITC/PI double staining combined with flow cytometry were used to measure the effect of STA on the apoptosis of TNBC cells. Western blot was employed to determine the expression changes of apoptosis-related proteins [cysteinyl aspartate-specific proteinase(caspase)-9, caspase-3, and poly-ADP-ribose polymerase(PARP)] and cancerous inhibitor of protein phosphatase 2A(CIP2A)/protein kinase B(AKT) signaling pathway proteins [CIP2A, AKT, phosphorylated(p)-AKT] in TNBC cells after STA treatment. To clarify the function of CIP2A in the action of STA, a CIP2A overexpression or CIP2A knockdown plasmid was transfected into cells, which were then treated with STA. Cell proliferation, apoptosis, and protein expression changes were evaluated by CCK-8, flow cytometry, and Western blot. Further, RT-qPCR and Western blot both showed that STA significantly downregulated the mRNA and protein levels of CIP2A and the protein level of c-Myc. The overexpression of c-Myc antagonized the downregulating effects of STA on the protein and mRNA levels of CIP2A, while the knockdown of c-Myc enhanced this effect. The drug affinity-responsive target stability(DARTS) assay and microscale thermophoresis(MST) assay confirmed the existence of direct binding between STA and c-Myc protein. The results indicated that STA significantly inhibited the proliferation and colony formation and induced the apoptosis of TNBC cells, manifested by an increased apoptosis rate, downregulated precursor protein expression of caspase-9 and caspase-3, and increased PARP cleavage. STA treatment reduced the p-AKT level but did not affect total AKT. Functionally, the knockdown of CIP2A enhanced the STA effects of inhibiting proliferation and inducing apoptosis, while the overexpression of CIP2A produced antagonistic effects. From a mechanism perspective, STA directly targets and binds to c-Myc to downregulate its expression, thereby inhibiting the transcription and translation of CIP2A and ultimately blocking the c-Myc/CIP2A signaling pathway. In conclusion, STA inhibits the malignant progression of TNBC by targeting the c-Myc/CIP2A signaling axis.

This study investigated the effects and molecular mechanisms of sarsasapogenin(SSG) on colorectal cancer(CRC). After HRT-18 and HCT116 cells were treated with varying concentrations of SSG, the effect of SSG on CRC cell proliferation, migration, and glycolysis was assessed by cell counting kit-8(CCK-8), real-time cellular analysis(RTCA), colony formation assay, high-content imaging analysis, and Seahorse glycolysis stress test. Changes in intracellular lactate release and adenosine triphosphate(ATP) levels under SSG treatment were measured by lactate and ATP detection kits, respectively. The effects of SSG on the transcription level and protein expression of the key glycolytic gene hypoxia-inducible factor 1α(HIF1α) were evaluated via quantitative polymerase chain reaction(qPCR) and Western blot. Direct binding between SSG and HIF1α was identified by drug affinity responsive target stability(DARTS). The UbiBrowser database, microscale thermophoresis(MST), and molecular docking simulations were employed to screen and validate the E3 ubiquitin ligase mediating HIF1α degradation. The results showed that SSG significantly inhibited CRC cell proliferation and colony formation in a dose-dependent manner. The 24-hour half-maximal inhibitory concentrations(IC_(50)) of SSG for HRT-18 and HCT116 cells were 6.275 μmol·L~(-1) and 7.029 μmol·L~(-1), respectively. After 24 hours of treatment with SSG at concentrations of 2.0, 4.0, and 6.0 μmol·L~(-1), the HRT-18 and HCT116 clone formation rate was significantly reduced. High-content imaging results showed that SSG significantly inhibited the migration ability of CRC cells. Seahorse glycolysis stress test, along with lactate and ATP measurements, demonstrated that SSG suppressed basal glycolysis, glycolytic capacity, glycolytic reserve, lactate release, and ATP content in CRC cells. The qPCR and Western blot results showed that SSG had no significant effect on HIF1α mRNA expression, and the HIF1α protein expression was downregulated. DARTS confirmed the direct binding of SSG to HIF1α. UbiBrowser analysis identified five E3 ubiquitin ligases potentially targeting HIF1α. Molecular docking and MST results indicated that the F-box/WD repeat-containing protein 7(FBXW7) exhibited strong binding affinity to HIF1α in the presence of SSG. In summary, SSG exerts its anti-cancer effects in CRC by targeting HIF1α to promote its FBXW7-mediated proteasomal degradation and consequently inhibiting glycolytic reprogramming.

This study investigates the inducing effects of jatrorrhizine(JAT), a bioactive compound extracted from Coptidis Rhizoma, on the p53-mediated ferroptosis in human colorectal cancer(CRC) cell lines HT29 and HCT116, specifically through the modulation of spermidine/spermine N1-acetyltransferase 1(SAT1). Cell viability was assessed via the cell counting kit-8(CCK-8) assay, while the colony formation assay was employed to determine the proliferative capacity. Cell cycle distribution was analyzed by flow cytometry, and mitochondrial membrane potential was evaluated via JC-1 dye. High-throughput RNA sequencing(RNA-seq) was performed to analyze transcriptomic changes in JAT-treated HT29 and HCT116 cells. Lentiviral transduction was utilized to generate SAT1-overexpressing HCT116 and HT29 cell lines, with transfection efficiency confirmed by qPCR. The levels of intracellular Fe~(2+), reactive oxygen species(ROS), malondialdehyde(MDA), and glutathione(GSH) were quantified via specific commercial assay kits. The protein and mRNA levels of key ferroptosis-related markers: solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long-chain family member 4(ACSL4), tumor protein p53, and SAT1 were determined by Western blot and qPCR, respectively. The results demonstrated that JAT treatment significantly inhibited both cell viability and proliferation, induced cell cycle arrest, and reduced mitochondrial membrane potential in HCT116 and HT29 cells. RNA-seq results revealed that the KEGG pathways most significantly enriched in JAT-treated cells were protein digestion and absorption, arginine and proline metabolism, glycine, serine and threonine metabolism, and ferroptosis. Notably, the mRNA level of SAT1 in the arginine metabolism pathway was significantly upregulated following JAT treatment. Furthermore, JAT treatment increased the levels of Fe~(2+), ROS, and MDA, while markedly decreasing GSH content in a dose-dependent manner. In addition, the treatment downregulated the protein levels of the ferroptosis inhibitors SLC7A11 and GPX4, and upregulated the expression of the pro-ferroptotic proteins ACSL4, SAT1, and p53. SAT1 overexpression inhibited cell proliferation and activated the p53-mediated ferroptosis signaling pathway. The combination of SAT1 overexpression and JAT treatment(oveSAT1+JAT group) exhibited a synergistic effect, showing superior efficacy over SAT1 overexpression alone(oveSAT1 group). In conclusion, JAT activates the p53-dependent ferroptosis signaling pathway by upregulating the key metabolic enzyme SAT1, which is involved in proline metabolism, thereby inhibiting colorectal cancer cell proliferation.

This study aimed to elucidate the mechanisms of diosgenin, a natural compound derived from traditional Chinese medicine, on the triple-negative breast cancer cell line MDA-MB-231, with a focus on its induction of cell cycle arrest and inhibition of cell migration. Multiple experimental approaches were employed, including RNA sequencing analysis, flow cytometry for cell cycle detection, wound healing assays to evaluate migration capacity, F-actin staining to observe cytoskeletal changes, transmission electron microscopy(TEM) to examine cellular morphology and filopodia alterations, as well as Western blot and immunofluorescence staining to detect the expression of related proteins. RNA sequencing revealed significant gene expression differences between the diosgenin-treated group and the control group, with KEGG and GO analyses showing enrichment of differentially expressed genes mainly in pathways related to cell cycle and migration. Flow cytometry demonstrated that diosgenin induced G_0/G_1 phase arrest in MDA-MB-231 cells in a dose-dependent manner. Further Western blot and immunofluorescence assays indicated that diosgenin regulated the cell cycle via the CDC25A-CDK2/4/6 axis. Wound healing assays showed that diosgenin significantly inhibited cell migration. F-actin staining indicated that diosgenin suppressed F-actin formation and disrupted its structure. TEM observations revealed a reduction in filopodia formation on the cell surface after diosgenin treatment. Moreover, Western blot and immunofluorescence results demonstrated that diosgenin inhibited migration by suppressing epithelial-mesenchymal transition(EMT) and the RhoA-ROCK1 signaling pathway. In conclusion, diosgenin induces G_0/G_1 phase arrest in MDA-MB-231 cells by regulating cell cycle-related proteins and significantly inhibits cell migration by suppressing F-actin formation, reducing filopodia on the cell surface, and inhibiting EMT and the RhoA-ROCK1 pathway.

This study aimed to elucidate the therapeutic effects and molecular mechanisms of Periplaneta americana extract(PAE) on ground-glass nodule(GGN)-type early-stage lung adenocarcinoma(LUAD) using patient-derived organoid(PDO) models. GGN-type patient-derived early-stage LUAD organoid(GGN-PDO) were successfully established. At the level of drug quality control and component analysis, the content of free amino acids and peptides in PAE were determined by high-performance liquid chromatography(HPLC), and the main components were further analyzed by liquid chromatography-mass spectrometry(LC-MS). Analysis of variance was used to evaluate the similarity between drugs of the same batch(Pearson correlation coefficient 0.87-0.99) and ensure quality control. At the histopathological level, hematoxylin-eosin(HE) staining and immunohistochemical(IHC) analysis of key molecular markers, including cytokeratin 7(CK7), thyroid transcription factor-1(TTF-1), and Ki-67, confirmed that the organoids recapitulated the tumor characteristics of parental tissues. Pharmacodynamic evaluation demonstrated that PAE significantly inhibited the proliferation and viability of GGN-PDOs in a dose-dependent manner, while immunofluorescence(IF) staining showed a marked reduction in the proliferation marker Ki-67. Flow cytometry further confirmed that PAE induced apoptosis in GGN-PDO, and Western blot analysis demonstrated that PAE downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax, thereby activating the apoptotic pathway. Notably, for the first time, the pro-apoptotic mechanism of PAE was shown to involve inhibition of the Janus kinase 1(JAK1)/phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) signaling pathway, as evidenced by markedly decreased protein levels of JAK1, PI3K, p-PI3K, AKT, and p-AKT following PAE treatment. In summary, this study is the first to use a clinically relevant GGN-PDO models to demonstrate that PAE induces apoptosis in early-stage LUAD via JAK1/PI3K/AKT pathway inhibition, providing a solid theoretical and experimental basis for developing innovative pharmacotherapies and non-surgical interventions against GGNs.

This study aims to explore the anti-gastric cancer mechanism of combined prescription of modified Liangfu and Jianpi Yiqi(CPMLJY) using a zebrafish xenograft model with gastric cancer, ultra-performance liquid chromatography(UPLC), and data-independent acquisition(DIA) proteomics. The active ingredients of CPMLJY were identified by UPLC. A zebrafish xenograft model was established by microinjecting human gastric cancer HGC-27 cells into the yolk sac of wild-type AB strain zebrafish. The maximum tolerated concentration(MTC) of CPMLJY was measured by a gradient dilution method. Zebrafish were randomly divided into normal control group, model control group, capecitabine group, and low-(1/4 MTC), medium-(1/2 MTC), and high-dose(MTC) groups of CPMLJY, with 30 zebrafish in each group. After 48 h at 35 ℃, the effect of CPMLJY on tumor proliferation and apoptosis was assessed via fluorescence labeling. Transgenic zebrafish(T-cell-red and vascular-green Fil-1 strains) were used for modelling to evaluate immune response and angiogenesis. DIA proteomics was used to reveal potential anti-gastric cancer mechanisms. The results showed that the main active ingredients of CPMLJY were flavonoids, steroidal saponins, and triterpenoid saponins. The medium-and high-dose of CPMLJY significantly inhibited tumor growth(P<0.05). High-dose of CPMLJY significantly increased the number of T cells, while medium-dose of CPMLJY significantly suppressed angiogenesis and induced apoptosis(P<0.05). Proteomics identified 143 upregulated and 233 downregulated proteins(P<0.05). CPMLJY exerted anti-tumor effects by modulating adipocyte lipolysis, forkhead box O(FoxO), wingless-related integration site(Wnt), and peroxisome proliferator-activated receptor(PPAR) signaling pathways, and amino acid metabolism(P<0.05). These findings support further clinical translation of CPMLJY for gastric cancer therapy.

This study aims to explore the mechanism of Shuangshen Yiwei Granules, an empirical prescription developed by Professor LU Zhi-zheng(a master of TCM), in regulating gastric acid secretion and impeding malignant progression of chronic atrophic gastritis with gastric intestinal metaplasia(GIM). Seventy-two specific-pathogen free(SPF) Wistar male rats were randomly divided into a normal group and a modeling group. GIM model rats were established by a four-factor GIM induction protocol involving "N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), ranitidine, irregular feeding, and sodium salicylate". After successful modeling, the rats in the modeling group were further divided into a model group, a positive control(Moluodan), and a Shuangshen Yiwei Granules group, with 12 rats in each group. Each group was continuously intervened for eight weeks. General conditions were observed, and pathological changes in the gastric mucosa were evaluated via hematoxylin-eosin staining. Serum pepsinogen Ⅰ(PGⅠ), pepsinogen Ⅱ(PGⅡ), and gastrin-17(G-17) were quantified by enzyme-linked immunosorbent assay. Parietal cell ultrastructure was analyzed via transmission electron microscopy. The apoptosis of gastric glandular cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining. Gastric pH was measured with precision test strips. The expression of apoptosis pathway-related proteins Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) and tumor necrosis factor receptor 1(TNFR1)/cysteine-aspartic acid protease-8(caspase-8) and acid secretion-related proteins cyclin dependent kinase 5(CDK5)/soluble N-ethylmaleimide sensitive factor attachment protein receptors(SNAREs) in gastric mucosal tissue were detected by Western blot. Compared to the normal group, the model group exhibited lethargy in mental state, pale eyes/ears/claws/tongue, tail desquamation, glandular atrophy of the gastric mucosa with disordered arrangement, and tumor-like structures, significantly reduced PGⅠ, PGⅠ/PGⅡ ratio, and G-17(P<0.01), significantly elevated PGⅡ(P<0.01), parietal cell nuclear condensation, loss and abnormal structures of mitochondria, significantly increased gastric glandular apoptosis(P<0.01), and significantly elevated gastric pH(P<0.01). The levels of p-JAK2/JAK2, p-STAT3/STAT3, B-cell lymphoma-2(Bcl-2)-associated X protein(Bax)/Bcl-2, TNFR1, TNFR1 associated via death domain(TRADD), Fas-associated death domain(FADD), and cleaved caspase-8/caspase-8 significantly increased(P<0.05, P<0.01), while histamine receptor H_2(HRH_2), CDK5, syntaxin 3(STX3), and synaptosome associated protein 25(SNAP25) significantly decreased(P<0.05). Compared with the model group, the Shuangshen Yiwei Granules group exhibited alleviated mental state, general conditions, and pathological features of the gastric mucosa, significantly upregulated PGⅠ, PGⅠ/PGⅡ ratio, and G-17(P<0.05, P<0.01), significantly downregulated PGⅡ(P<0.01), ameliorated parietal cell ultrastructure to varying degrees, significantly reduced gastric glandular apoptosis(P<0.01), significantly lowered gastric pH(P<0.01), significantly decreased expression of p-JAK2/JAK2, p-STAT3/STAT3, Bax/Bcl-2, TNFR1, TRADD, FADD, and cleaved caspase-8/caspase-8(P<0.05, P<0.01), and significantly increased expression of HRH_2, CDK5, STX3, and SNAP25(P<0.05). To sum up, Shuangshen Yiwei Granules potentially inhibit JAK2/STAT3 and TNFR1/caspase-8 signaling pathways to suppress gastric glandular apoptosis while activating CDK5/SNAREs to enhance parietal cell acid secretion, thereby restoring gastric acid homeostasis and blocking GIM progression.

Chronic gastritis, a chronic and insidious inflammatory condition of the gastric mucosa, is characterized by nonspecific early symptoms and a high risk of progression to gastric cancer. Persistent inflammatory stimulation accelerates the "inflammation-cancer transformation" by remodeling the microenvironment, and suppressing this process is critical for gastric cancer prevention and treatment. Nuclear factor-κB(NF-κB) serves not only as a central hub for inflammatory regulation but also as a key node connecting pathways such as Toll-like receptor 4(TLR4), NOD-like receptor protein 3(NLRP3), mitogen-activated protein kinase(MAPK), signal transducer and activator of transcription 3(STAT3), and protein kinase B(AKT). Extensive studies have demonstrated that monomers and herbal formulas in traditional Chinese medicine(TCM) can modulate NF-κB-related signaling pathways to mitigate the progression of "inflammation-cancer transformation" of chronic gastritis, achieving multi-level, multi-target intervention in gastric cancer. Based on this, this article systematically reviewed the dynamic network of NF-κB and relevant crosstalk pathways in "inflammation-cancer transformation" and the mechanisms of TCM-based interventions, providing theoretical foundations for optimizing clinical strategies and advancing gastric cancer prevention.

Mantle cell lymphoma (MCL) is a rare B-cell lymphoma characterized by both the incurable nature of indolent lymphomas and the clinical course of aggressive lymphomas. The integration of high-dose cytosine arabinoside (Ara-C) and autologous hematopoietic stem cell transplantation (ASCT) has led to substantial improvement in the outcomes of MCL patients in the immunochemotherapy era. More recently, the widespread use of small molecule targeted agents, particularly Bruton tyrosine kinase inhibitor (BTKi), has re-shaped the therapeutic landscape of MCL patients and challenged the traditional role of high-dose Ara-C and ASCT. Novel immunotherapies including bi-specific antibodies and chimeric antigen receptor T-cell (CAR-T) therapy have emerged as important treatment options for MCL patients with relapsed or refractory disease. With advances in multi-omics profiling, the development of personalized, potentially curative strategies based on individual genetic and immune features is expected to become a major focus of future research on MCL. This article will delve into the latest research progress in the treatment and genetics and translational research on MCL patients, focusing on the latest progress of research on the treatment of newly diagnosed MCL patients, treatment of relapsed/refractory MCL patients, and the genetics and translational treatment of MCL patients, and explore the evolution and future direction of its treatment model.

Objective: To investigate the clinicopathological features of primary pulmonary epithelioid hemangioendothelioma (PEHE). Method: Forty cases of PEHE were diagnosed from October 2010 to June 2024 at the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. A retrospective analysis was conducted on their histological features, imaging findings, immunohistochemical characteristics and molecular phenotypes. Subsequently, the clinicopathological features were summarized. The patients were followed up. Result: Of the 40 cases, there were 19 males and 21 females, age 52.5 (43.0, 62.0) years old. Most patients were admitted for respiratory symptoms, mainly cough (25/40) and expectoration (14/40). Computed tomography findings mainly showed multiple intrapulmonary nodules (33/40) and solitary nodules in 7 cases (7/40). Tumor maximum diameters ranged from 3 to 70 mm, with a median of 19 (12, 35) mm. Grossly, all lesions appeared as grayish-white nodules with ill-defined margins and mucoid cut surfaces. Microscopically, tumor cells showed centrifugal distribution around blood vessels, arranged in irregular nests; local mucoid degeneration and chondroid matrix were noted. Intracytoplasmic vacuoles with red blood cells were noted in some tumor cells, indicating primitive vascular lumen differentiation. At the molecular level, WWTR1-CAMTA1 gene fusion was identified in 36 cases and YAP1-TFE3 fusion in 4 cases. Immunohistochemical results showed diffuse positivity for CD31 (38/38), CD34 (36/40), ERG (40/40) and Fli-1 (40/40), and focal positivity for TFE-3 (4/34). Therapeutic responses of 40 patients were assessed using the Response Evaluation Criteria in Solid Tumors criteria: complete response in 4 cases (10.0%), partial response in 5 (12.5%), stable disease in 7 (17.5%), and progressive disease in 24 (60.0%). Conclusions: PEHE is a rare vascular-derived tumor, radiologically characterized by multiple bilateral pulmonary nodules. It has non-specific clinical manifestations; combined use of highly sensitive and specific endothelial markers and genetic testing helps reach the definitive diagnosis. PEHE has an overall indolent course, with long-term survival in some patients. However, multiple lesions, pleural invasion, and distant metastasis may be linked to worse prognoses.

Objective: To score pheochromocytoma and paraganglioma (PPGL) by using the composite pheochromocytoma and paraganglioma grading system (COPPS), to analyze the correlations of COPPS, the pheochromocytoma of the adrenal gland scaled score (PASS), and the grading system for adrenal pheochromocytoma and paraganglioma (GAPP) with tumor metastasis or recurrence and to explore the relationship between gene mutations and metastasis or recurrence in some PPGL. Methods: Clinicopathological data of the 186 paragangliomas diagnosed from January 2012 to December 2022 at Beijing Friendship Hospital, Beijing, China, the Peking University Third Hospital, Beijing, China, and the First Affiliated Hospital of Shihezi University, Shihezi, China were collected and analyzed. Predictive values of the three systems for tumor metastasis or recurrence were evaluated. Immunohistochemistry was performed using the EnVision staining method. Whole-exome sequencing was used to detect gene mutations in 15 tumors. Results: Among the 186 PPGL patients, there were 93 females and 93 males, age 49 (47, 50) years old. Metastasis or recurrence occurred in 60 cases. 34 of the tumors were located in the retroperitoneum. The maximum tumor diameter was >7 cm in 42 cases. A COPPS score ≥3 was observed in 97 cases (52.2%, 97/186), among whom 53 cases (54.6%, 53/97) experienced metastasis or recurrence. The metastasis/recurrence rate in the COPPS score≥3 group was significantly higher than that in the <3 group (χ2=46.469,P<0.001). Tumor location in the retroperitoneum, presence of large nests of cells, pathological mitosis, spindle cells, capsular invasion, and fat infiltration were all associated with a COPPS score ≥3 (χ2=18.370, 51.730, 8.914,18.750, 62.481, 19.354, all P<0.05). The sensitivity of COPPS for predicting metastasis/recurrence was 88.3% (53/60), while the specificity was 65.1% (82/126). Negative expression of SDHB was observed in 50 cases, and negative expression of S-100 protein was observed in 96 cases. DNA extraction failed to produce qualified DNA in 3 cases; among the remaining 12 tumors that were subject to DNA extraction, 739 mutations (in 658 genes) were detected, including 300 germline mutations (in 265 genes) and 439 somatic mutations (in 408 genes). Related pathogenic germline mutation genes occurred on: SDHA, MDH2, MEN1, EGLN1, RET and SDHB. All 12 patients had more than four pathogenic germline mutations. Somatic pathogenic mutation genes included ATRX, KIF1B, EPAS1, HRAS, NF1, and MAML3. Conclusions: A higher COPPS score (≥3 versus <3) is associated with a higher metastasis/recurrence rate. Its sensitivity for predicting tumor metastasis/recurrence is higher than that of GAPP, while its specificity is higher than that of PASS. The combined use of negative SDHB and S-100 protein expression with COPPS could be used to stratify the risk of metastasis/recurrence in PPGL.

Objective: To investigate the clinicopathological and molecular genetic features of micronodular thymoma (MNT) and micronodular thymic carcinoma (MNC) with lymphoid stroma. Methods: Seventeen cases of micronodular neoplasms with lymphoid stroma diagnosed at the Third Affiliated Hospital of Soochow University, Changzhou, China from March 2017 to October 2024 were collected (16 cases of MNT and 1 case of MNC). Their clinicopathological data were reviewed, and their paraffin-embedded sections were stained with immunohistochemistry (IHC). Three cases of MNT and one case of MNC were also examined using next generation sequencing (NGS). Results: Of the 17 patients with MNT or MNC, 7 were male and 10 were female, age 66 (63, 72)years old. Microscopically, tumor cells were scattered in the lymphoid stroma as solid epithelioid nests. The tumor nests were relatively regular in 16 cases, and the tumor cells were mainly short spindle or oval, with bland morphology. Tumor cells of MNC showed marked cytological atypia, conspicuous nucleoli, frequent mitotic figures, and fibrous stroma. IHC showed that tumor cells of all 17 cases diffusely expressed CKpan, CK19 and CK5/6. CD5, CD117 and Glut1 were diffusely positive in the MNC case, but negative in any of the 16 MNT cases. In contrast, there were a considerable number of immature T lymphocytes positive for CD99, TdT and CD1a in the lymphoid stroma around MNT tumor nodules, but not in the MNC. Among the 3 MNT cases subjected to NGS testing, 2 cases had missense mutations in GTF2I (p.L424H), and 1 had missense mutations in HRAS (p.G13V and p.L120P). In the MNC case subjected to NGS testing, gene alterations such as a frameshift mutation in BRCA2 (p.D1451Ifs*12) and a missense mutation in TP53 (p.I195T) were detected, but not GTF2I gene mutations. Conclusions: MNC cells are more atypical than MNT cells. CD5, CD117 and Glut1 are diffusely expressed in MNC, but not in MNT. The genetic alterations in MNC are more diverse than those of MNT, but MNC lacks the characteristic GTF2I mutation of MNT.

Objective: To investigate the prognostic stratification value of MLH1 promoter methylation in a mismatch repair deficiency (dMMR)-type endometrioid endometrial carcinoma (EEC). Methods: A total of 338 patients with confirmed diagnosis of dMMR EEC at Third Hospital of Peking University Health Science Center, from July 2005 to June 2023 were analyzed. Based on the promoter methylation, they were classified into a dMMR methylated (dMMR MET) group (177 cases) and a dMMR nonmethylated (dMMR nonMET) group (127 cases). Somatic mutations were analyzed by targeted sequencing (196/425-gene panel), and transcriptomic differences were assessed by RNA sequencing (Master panel). We compared the clinicopathological characteristics, gene mutation/expression profiles, and molecular pathway activities systematically between the two groups. Results: Compared with the dMMR nonMET group, patients of the dMMR MET group were older significantly [(56.89±8.85) vs. (53.76±9.45) years, P=0.003] and had tumor size of larger diameters [(3.39±1.78) vs. (2.71±1.31) cm, P=0.014]. The menopausal proportion (66.9% vs. 48.8%, P=0.002) and the proportion with tumor buddings (47.5% vs. 30.4%, P=0.036) were higher. No significant differences were identified in FIGO stage, histologic grade, depth of myometrial invasion, lymphovascular invasion, or rate of lymph node metastasis (P>0.05). Mutational profiling revealed that the nonMET group had significantly higher mutation frequencies in CHD4, NF1, SMARCA4, and RET (P<0.05). Transcriptomic analysis demonstrated upregulation of immune-related genes (CCL21, CXCL2) in the MET group, and downregulation of epithelial-mesenchymal transition (EMT)-associated genes (SOX2, FOXA1). Both GO and KEGG enrichment analyses of different gene expression in the MET group demonstrated an association with the MAPK pathway. However, Hallmark pathway analysis showed no significant differences in overall pathway activity between the two groups. Survival analysis revealed no significant differences in progression-free survival (P=0.206) or overall survival (P=0.813) between the groups. Conclusions: The methylation status of the MLH1 promoter has limited value in predicting the prognosis of dMMR EEC. Molecular pathways heterogeneity between the methylated and nonmethylated subgroups suggests the necessity of integrate multi-dimensional indicators to optimize stratification strategies, instead of relying on a single epigenetic marker.