Tumor Cell Clusters Research Articles

An overview of invasive micropapillary carcinoma of the breast: past, present, and future

Invasive micropapillary carcinoma of the breast (IMPC) exhibits a unique micropapillary structure and “inside-out” growth pattern. Despite its extremely low incidence, IMPC has attracted considerable attention owing to its poor prognosis. Since Siriaunkgul and Tavassoli first proposed the term IMPC in 1993 to describe its morphological characteristics, with tumor cell clusters arranged in a pseudopapillary structure within the glandular cavity, its diagnostic rate has substantially increased. Based on the in-depth study of IMPC, a more comprehensive understanding of its epidemiology, clinicopathological features, and diagnostic criteria has been achieved in recent years. The pathogenesis and specific therapeutic targets of IMPC remain unclear. However, numerous studies have delved into its high-risk biological behavior. This review discusses the opportunities and challenges associated with IMPC.

Abstract A055: Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.

BIOM-62. CIRCULATING TUMOR CELLS IN MEDULLOBLASTOMA: A NOVEL PERIPHERAL BIOMARKER FOR CNS DISEASE

Abstract BACKGROUND Medulloblastoma is an embryonal tumor of the cerebellum and the most common pediatric malignancy of the central nervous system (CNS). Diagnosis, staging, and disease monitoring require invasive neurosurgical biopsy or MRI. No blood or cerebrospinal fluid (CSF) biomarkers exist. We demonstrate the presence of circulating tumor cell clusters (CTCCs) in patients with medulloblastoma and use a transcriptomic approach to describe their gene expression. Design/Method: We enrolled 34 total patients in a longitudinal study: 24 with medulloblastoma and 10 without malignancy. CTCCs are captured from unprocessed whole blood using a physics-based label-free approach to CTCC isolation. RNA sequencing was performed on 21 samples at time of diagnostic resection from 6 patients with medulloblastoma. Sequencing reads were assessed for quality control, trimmed, and aligned to the human genome. Differential gene expression (DGE) was performed against whole blood from healthy controls. Reactome pathway database of curated, peer-reviewed gene sets are used for gene set enrichment analysis (GSEA). RESULTS The presence of peripherally CTCC patients with medulloblastoma is not previously known and was detected in all patients with medulloblastoma whereas none were observed from patients with non-malignant hematological conditions. RNA-sequencing demonstrate separation on principal component analysis and gene-expression heatmap against whole blood healthy control. DGE shows upregulation of genes indicating stem-like state. GSEA show enrichment of medulloblastoma molecular pathways and gene sets defining integrin, extracellular matrix remodeling, and cell adhesion pathways. Transient elevation of peripheral CTCC followed by return to baseline levels was associated with durable disease response to therapy, whereas persistence or elevation of CTCC in CSF correlated with disease recurrence. CONCLUSION The universal and specific finding of peripherally captured CTCC arising from parent tumor in the CNS within our patient cohort suggests a utility as a tumor biomarker. Future sequencing is planned comparing CTCC transcriptome against parent tumor.

Prognostic value of isolated tumor cells and micrometastasis of lymph nodes in invasive urinary bladder cancer.

The prognostic significance of nodal micrometastasis and isolated tumor cells (ITC) in urinary bladder cancer (UBC) is unknown. We aimed to evaluate the prevalence, clinical impact, and clinicopathological characteristics of nodal micrometastasis and ITC in UBC. A total of 124 patients with UBC undergoing surgery were investigated. Detection of micrometastasis and ITC was performed using pancytokeratin immunohistochemistry (IHC). Histopathologic and clinical findings were correlated with patients' outcome. IHC detected nodal micrometastasis and ITC (pNmi group) in 12.9% (13/101) of originally node-negative patients and in 26.1% (6/23) of originally node-positive patients (pN+ group). The remaining 88 were truly node-negative patients (pN0 group). After IHC, all 13 patients in the pNmi group were upstaged from pN0 to pN1-2 and one patient in the pN+ group was changed from pN1 to pN2. Nodal micrometastasis and ITC were significantly associated with mixed urothelial carcinoma (UC) (p = 0.002), UC with discohesive pattern (p = 0.006), glandular differentiation (p = 0.043), lymphovascular invasion (p = 0.009), and budding-like tumor cell clusters (p = 0.002). The pNmi group had significantly worse cancer-specific survival than the pN0 group in univariate (p = 0.004) and multivariate (p = 0.040) analysis. IHC frequently identified nodal micrometastasis and ITC in originally node-negative UBC patients on routine pathological examination. Nodal micrometastasis and ITC were independently associated with cancer-related mortality in UBC. IHC might be selectively used to detect micrometastasis and ITC in UBC having specific pathological features.

Evaluation of Vincamine Loaded with Silver Nanoparticles as a New Potential Therapeutic Agent Against Ehrlich's Solid Carcinoma in Mice.

Vincamine, a monoterpenoid indole alkaloid with vasodilatory properties, is extracted from the leaves of Vinca minor. The present study aimed to determine the potential anticancer effects of vincamine loaded in silver nanoparticles (VCN-AgNPs) in mice with Ehrlich solid carcinoma (ESC). After tumor transplantation, the mice were divided into five groups: ESC, ESC+Cisplatin (CPN; 5 mg/kg), ESC+VCN (40 mg/kg), ESC+AgNPs (6 mg/kg), and ESC+VCN-AgNPs (20 mg/kg). The administration of VCN-AgNPs to ESC-bearing mice improved their survival rate and reduced their body weight, tumor size, and tumor weight compared to the ESC group. Furthermore, VCN-AgNPs intensified oxidative stress in tumor tissues, as evidenced by elevated levels of lipid peroxidation (LPO) and nitric oxide (NO), along with a reduction in the levels of the antioxidants investigated (GSH, GPx, GR, SOD, CAT, and TAC). Furthermore, VCN-AgNPs increased the apoptotic proteins Bax and caspase-3, decreased the anti-apoptotic protein (Bcl-2), increased the inflammatory markers TNF-α and IL-1β, and inhibited angiogenesis by lowering VEGF levels in tumor tissues, all of which led to apoptosis. Furthermore, histopathological studies showed that VCN-AgNPs suppressed the progression of Ehrlich carcinoma and induced the formation of clusters of necrotic and fragmented tumor cells. VCN-AgNPs possess cytotoxic and genotoxic effects against ESC because of their pro-oxidant, pro-apoptotic, pro-inflammatory, and antiangiogenic effects. Additionally, the combination of VCN-AgNPs was more effective and safer than chemically synthesized AgNPs, as indicated by an increase in the lifespan of animals and the total tumor inhibition index.

Atypical Gastric Glomus Tumor of Uncertain Malignant Potential in A 16-Year-Old Female; A Rare Entity with Unique Findings

Abstract Introduction/Objective Gastric glomus tumor (GGT) is a rare mesenchymal tumor that is challenging to diagnose, especially when the immunoprofile overlaps with other more common gastric mesenchymal tumors. The majority of GGT are benign, however, rarely it can be atypical or even malignant. Herein, we present a case of a young female with atypical gastric glomus tumor of uncertain malignant potential. Methods/Case Report A 16-year-old female presented to the emergency with abdominal pain, coffee ground emesis, and melena. Imaging showed a 3.2 cm well-defined gastric mass arising from the muscularis propria. Results (if a Case Study enter NA) The FNA biopsy revealed clusters of tumor cells with uniform characteristics, exhibiting distinct cell membranes and eosinophilic to clear cytoplasm, surrounding prominent vasculature. Immunohistochemical analysis showed that the tumor cells were positive for SMA, synaptophysin, vimentin, calponin, and caldesmon, with weak, patchy positivity for DOG1. CD34 highlighted the vessels separating the tumor cellular clusters into lobules. CD117 and Desmin were negative. The Ki67 proliferation index varied with hotspot foci 25-30%. Morphology and immunoprofile suggest glomus tumor, however, neuroendocrine tumor and epithelioid GIST needed to be excluded. Subsequent resection specimen showed that the tumor was located within the muscularis propria with extension into the serosal vessels, suggesting vascular invasion, along with foci of cellular spindling and nuclear irregularity are identified. No necrosis noted. Tumor cells were positive for cyclin D1 while negative for myogenin, chromogranin, and DOG1. Overall, findings were consistent with GGT of uncertain malignant potential. Conclusion Although GGT are rare, they should be considered in the differential diagnosis of gastric mesenchymal tumors. The presence of diffuse SMA staining can help distinguishing them from GIST. It is crucial to scrutinize for signs of cellular and nuclear atypia, particularly in deeply seated glomus tumors.

Local Invasion Patterns Characterized by SARIFA and Tumor Budding Differ and Have Distinct Prognostic Significance in Esophageal Adenocarcinoma and Squamous Cell Carcinoma.

Both esophageal squamous cell carcinoma (ESQCC) and adenocarcinoma (EAC) are known to have poor prognosis. We aimed to investigate the invasion front areas of 57 ESQCC and 43 EAC cases to find histological signs of metastatic progression. Tumor cell clusters with different cell counts, including tumor buds (TBs) and poorly differentiated clusters (PDCs), were assessed. The presence of the recently described Stroma AReactive Invasion Front Area (SARIFA) phenomenon, which defines a direct contact between tumor cells and adipocytes, was more frequently observed in EAC than in ESQCC (p = 0.004). In adenocarcinomas, a higher prevalence of SARIFA was observed in tumors with a higher number of small clusters (TBs and small PDCs; p < 0.001); furthermore, both the high number of TBs (p = 0.016) and the presence of SARIFA (p = 0.001) correlated with a higher pT stage. SARIFA positivity in EAC (p = 0.011) and high TB in ESQCC (p = 0.0006) were found to be independent prognostic factors for lymph node metastases. Moreover, in ESQCC, the higher absolute number of both TBs and PDCs was associated with shorter overall survival (p = 0.0269 and p = 0.0377, respectively). Our results suggest that the histological subtypes of esophageal cancer behave differently, namely, that different features of the invasion front are of prognostic significance.

Exploring the Usefulness of Tumor Budding as a Histopathological Marker Compared to Tumor-Node-Metastasis (TNM) Staging in Breast Cancer.

Tumor budding, defined as small clusters of tumor cells at the invasive front of carcinomas, has gained attention as a potential prognostic marker in various cancers. This study aimed to evaluate the utility of tumor budding as a histopathological marker in breast cancer and compare it to traditional prognostic markers such as histological grading, tumor-node-metastasis (TNM) staging, and molecular subtypes. A prospective, cross-sectional study was conducted over two years (June 2022 to May 2024) in the Department of Pathology at Jawaharlal Nehru Medical College, Wardha. Seventy-two female patients diagnosed with breast carcinoma who underwent modified radical mastectomy were included. Tumor budding was assessed through histopathological analysis and categorized as high or low. Statistical correlations were established between tumor budding and tumor size, histological grade, lymph node involvement, vascular invasion, and molecular subtypes (estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and triple-negative breast cancer, TNBC). The chi-square test and multiple regression analyses were applied to assess significance. High tumor budding was observed in 68.1% of patients and was significantly associated with larger tumor size (p = 0.040), higher histological grade (p = 0.009), lymph node metastasis (p = 0.002), and vascular invasion (p = 0.003). The postmenopausal age group (>55 years) demonstrated a higher prevalence of high budding (p = 0.010). No significant correlation was found between tumor budding and molecular subtypes (p = 0.39), although high budding was more frequent in TNBCcases. Tumor budding is significantly associated with more aggressive tumor characteristics in breast cancer. Incorporating tumor budding into routine pathological assessments alongside TNM staging and histological grading may enhance the ability to identify high-risk patients and guide treatment strategies. Further large-scale, multicenter studies are warranted to confirm its prognostic value.

32P Patient derived circulating tumor cell clusters for personalized chemotherapy

32P Patient derived circulating tumor cell clusters for personalized chemotherapy

In vitro evidence for the potential of EGFR inhibitors to decrease the TGF-β1-induced dispersal of circulating tumour cell clusters mediated by EGFR overexpression

Most cancer-related deaths are due to the spread of tumour cells throughout the body—a process known as metastasis. While in the vasculature, these cells are referred to as circulating tumour cells (CTCs) and can be found as either single cells or clusters of cells (often including platelets), with the latter having the highest metastatic potential. However, the biology of CTC clusters is poorly understood, and there are no therapies that specifically target them. We previously developed an in vitro model system for CTC clusters and proposed a new extravasation model that involves cluster dissociation, adherence, and single-cell invasion in response to TGF-β1 released by platelets. Here, we investigated TGF-β1-induced gene expression changes in this model, focusing on genes for which targeted drugs are available. In addition to the upregulation of the TGF-β1 signalling pathway, we found that (i) genes in the EGF/EGFR pathway, including those coding for EGFR and several EGFR ligands, were also induced, and (ii) Erlotinib and Osimertinib, two therapeutic EGFR/tyrosine kinase inhibitors, decreased the TGF-β1-induced adherence and invasion of the CTC cluster-like line despite the line expressing wild-type EGFR. Overall, we suggest that EGFR inhibitors have the potential to decrease the dispersal of CTC clusters that respond to TGF-β1 and overexpress EGFR (irrespective of its status) and thus could improve patient survival.

Label-Free Microfluidic Apheresis of Circulating Tumor Cell Clusters.

Screening liters of blood (i.e., apheresis) represents a generalized approach to promote the reliable access to circulating tumor cell clusters (CTCCs), which are known to be highly metastasis-competent, yet ultrarare. However, no existing CTCC sorting technology has demonstrated high throughput, high yield, low shear stress, and minimal blood dilution simultaneously as required in apheresis. Here, a label-free method is introduced termed Precision Apheresis for Non-invasive Debulking of cell Aggregates (PANDA) to continuously isolate CTCCs from undiluted blood to clean buffer through size sorting, processing 1.4 billion cells per second. The cell focusing is optimized within whole blood leveraging secondary transverse flow and margination. The PANDA chip recovers >90% of spiked ≈24 rare HeLa cell clusters from 100mL undiluted blood samples (equivalent to ≈500 billion blood cells) at 1 Lh-1 throughput, with ≤20s device residence time, ≤15Pa shear stress, and >99.9% return of blood components. The technology lays the groundwork for future routine isolation to increase the recovery of these ultrarare yet clinically significant tumor cell populations from large volumes of blood to advance cancer research, early detection, and treatment.

Metal complex lipid-based nanoparticles deliver metabolism-regulating lomitapide to overcome CTC immune evasion via activating STING pathway

Activating the cGAS-STING pathway of circulating tumor cell clusters (CTC clusters) represents a promising strategy to mitigate metastases. To fully exploit the potential of cholesterol-regulating agents in activating CTCs’ STING levels, we developed a nanoparticle (NP) composed of metal complex lipid (MCL). This design includes MCL-miriplatin to increase NP stiffness and loads lomitapide (lomi) modulating cholesterol levels, resulting in the creation of PLTs@Pt-lipid@lomi NPs. MCL-miriplatin not only enhances lomi’s eliciting efficacy on STING pathway but also increases NPs’ stiffness, thus a vital factor affecting the penetration into CTC clusters to further boost lomi’s ability. Demonstrated by cy5 tracking experiments, PLTs@Pt-lipid@lomi NPs quickly attach to cancer cell via platelet membrane anchorage, penetrate deep into the spheres, and reach the subcellular endoplasmic reticulum where lomi regulates cholesterol. Additionally, these NPs have been shown to track CTCs in the bloodstream, a capability not demonstrated by the free drug. PLTs@Pt-lipid@lomi NPs more efficiently activate the STING pathway and reduce CTC stemness compared to free lomi. Ultimately, PLTs@Pt-lipid@lomi NPs reduce metastasis in a post-surgery animal model. While cholesterol-regulating agents are limited in efficacy when being repositioned as immunomodulatory agents, this MCL-composing NP strategy demonstrates the potential to effectively deliver these agents to target CTC clusters.

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Spread Through Air Spaces: Interresponder Agreement and Comparison Between Pulmonary and General Pathologists

Spread through air spaces (STAS), an important prognostic indicator included in the 2015 World Health Organization classification, is defined as micropapillary, solid, and/or single tumor cell clusters beyond the edge of the main mass and distinct from processing artifacts. This study aimed to assess the interresponder agreement on current STAS criteria vs artifacts, identify discrepancies, and compare responses between pulmonary and general pathologists. A multiple-choice online questionnaire illustrating multiple criteria for STAS vs artifacts was available internationally for 6 days to Pulmonary Pathology Society members, thoracic pathology course attendees, and International Association for the Study of Lung Cancer pathology committee members. Additional 4 questions gathered demographic and practice setting information. One hundred thirty-six unique responses were analyzed. The majority were from North America and Europe (42.6% and 30.2%), practicing pulmonary pathology (70.6%) in academia (64.7%), and with >20 years of experience (31.6%). Excluding trainees, the greatest overall agreement was in defining solid and micropapillary tumor clusters of STAS located ≥3 alveolar spaces from the main tumor edge (91.5%) and recognizing strips of ciliated cells as artifacts (97.7%). Lesser agreement on STAS was evident when tumor cell clusters were immediately adjacent to the tumor edge, a single tumor cell cluster was present at the tissue edge, tumor cell clusters were jagged edged, or tumor cell clusters were admixed with ciliated cell strips (artifacts). There was no significant difference in agreements on STAS for multiple criteria between pulmonary and general pathologists. Significant interresponder agreement on STAS vs artifacts was achieved only for a few criteria. To improve the reproducibility of STAS vs artifacts, areas of lesser agreement require further clarification.

Fibrinolytic Platelet Decoys Reduce Cancer Metastasis by Dissociating Circulating Tumor Cell Clusters.

During metastasis, circulating tumor cells (CTCs) can travel in the bloodstream as individual cells or clusters, associated with fibrin and platelets. Clusters have a higher metastatic potential due to their increased ability to withstand shear stress and arrest in small vessels. Moreover, CTC-platelet interaction protects CTCs from shear stress and immune detection. The objective of this project is to develop a fibrinolytic platelet system to leverage platelet-CTC interactions and dissociate CTC clusters. For this approach, tissue plasminogen activator (tPA) is loaded onto two modified platelet systems: platelet Decoys and lyophilized platelets. The activities of the systems are characterized using a Förster Resonance Energy Transfer-based assay and an angiogenic assay. Furthermore, the ability of the system to dissociate cancer cell clusters in vitro is assessed using light transmission aggregometry. The data demonstrates that the fibrinolytic platelets can maintain tPA activity, interact with CTCs, and dissociate cancer cell clusters. Finally, fibrinolytic platelets are assessed in vivo, demonstrating a decreased tumor load and increased survival with tPA-Decoy treatment, which is selected as the optimal treatment based on favorable in vitro results and in vivo trials. Therefore, this fibrinolytic platelet approach is a promising method for leveraging platelet-CTC interactions to disperse CTC clusters and reduce metastasis.

CHAI: consensus clustering through similarity matrix integration for cell-type identification.

Several methods have been developed to computationally predict cell-types for single cell RNA sequencing (scRNAseq) data. As methods are developed, a common problem for investigators has been identifying the best method they should apply to their specific use-case. To address this challenge, we present CHAI (consensus Clustering tHrough similArIty matrix integratIon for single cell-type identification), a wisdom of crowds approach for scRNAseq clustering. CHAI presents two competing methods which aggregate the clustering results from seven state-of-the-art clustering methods: CHAI-AvgSim and CHAI-SNF. CHAI-AvgSim and CHAI-SNF demonstrate superior performance across several benchmarking datasets. Furthermore, both CHAI methods outperform the most recent consensus clustering method, SAME-clustering. We demonstrate CHAI's practical use case by identifying a leader tumor cell cluster enriched with CDH3. CHAI provides a platform for multiomic integration, and we demonstrate CHAI-SNF to have improved performance when including spatial transcriptomics data. CHAI overcomes previous limitations by incorporating the most recent and top performing scRNAseq clustering algorithms into the aggregation framework. It is also an intuitive and easily customizable R package where users may add their own clustering methods to the pipeline, or down-select just the ones they want to use for the clustering aggregation. This ensures that as more advanced clustering algorithms are developed, CHAI will remain useful to the community as a generalized framework. CHAI is available as an open source R package on GitHub: https://github.com/lodimk2/chai.

Neuroendocrine Lesions Arising From Mediastinal Teratoma-A Case Report and Literature Review.

Background. Neuroendocrine lesions arising from mediastinal teratomas are rare tumors with only small number of patients reported in literature. The behavior of these lesions appears to be different from traditional neuroendocrine neoplasms. A comprehensive review will be valuable for histologic assessment and treatment planning for similar cases. Case presentation. We present an example of a 57-year-old man who presented with cough. Subsequent work-up revealed an anterior mediastinal mass of 2.1 cm on computed tomography. The patient underwent robot-assisted thoracoscopic thymectomy. Histological examination revealed a mature cystic teratoma with a neuroendocrine component consisting of clusters of tumor cells with round to oval nuclei and a "salt-and-pepper" chromatin pattern. The tumor cells were immunoreactive to cytokeratin, synaptophysin, chromogranin, and INSM1, with a Ki-67 proliferative index of 4%. A histological diagnosis was mature teratoma with well-differentiated low-grade neuroendocrine tumor (carcinoid) was made. The patient was well and without disease after complete surgical excision at 10 months. Literature review. Literature reviewed yielded 13 examples of neuroendocrine lesions arising from mediastinal teratomas. No disease-related mortality was reported, even in lesions with high-grade neuroendocrine, carcinomatous, or immature teratomatous components. Conclusions. Surgical removal is the mainstay of treatment of these lesions, and the presence of a neuroendocrine component does not appear to negatively affect prognosis.

Integrating machine learning and single-cell analysis to uncover lung adenocarcinoma progression and prognostic biomarkers.

The progression of lung adenocarcinoma (LUAD) from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (IAC) involves a complex evolution of tumour cell clusters, the mechanisms of which remain largely unknown. By integrating single-cell datasets and using inferCNV, we identified and analysed tumour cell clusters to explore their heterogeneity and changes in abundance throughout LUAD progression. We applied gene set variation analysis (GSVA), pseudotime analysis, scMetabolism, and Cytotrace scores to study biological functions, metabolic profiles and stemness traits. A predictive model for prognosis, based on key cluster marker genes, was developed using CoxBoost and plsRcox (CPM), and validated across multiple cohorts for its prognostic prediction capabilities, tumour microenvironment characterization, mutation landscape and immunotherapy response. We identified nine distinct tumour cell clusters, with Cluster 6 indicating an early developmental stage, high stemness and proliferative potential. The abundance of Clusters 0 and 6 increased from AAH to IAC, correlating with prognosis. The CPM model effectively distinguished prognosis in immunotherapy cohorts and predicted genomic alterations, chemotherapy drug sensitivity, and immunotherapy responsiveness. Key gene S100A16 in the CPM model was validated as an oncogene, enhancing LUAD cell proliferation, invasion and migration. The CPM model emerges as a novel biomarker for predicting prognosis and immunotherapy response in LUAD patients, with S100A16 identified as a potential therapeutic target.

The Prognostic Value of Tumor Cell Clusters in the Fallopian Tube Lumen in Stage I Endometrioid Carcinoma.

The aim of this study was to investigate the pathologic prognostic factors such as tumor cell clusters (TCCs) in the fallopian tube lumen, myometrial invasion patterns, and positive peritoneal cytology (PPC) in women with Stage I endometrial endometrioid carcinoma (EEC). From 2009 to 2020, consecutive patients with Stage I EEC who underwent hysterectomy and bilateral salpingectomy were included. The primary outcome was the recurrence-free survival (RFS) rate, and the clinicopathological factors affecting RFS were analyzed. A total of 765 patients were enrolled. Seventeen patients (2.2%) had TCC in the fallopian tube lumen, and 58 patients showed a microcystic elongated and fragmented pattern (7.6%). PPC was found in 19 patients (2.5%). The median follow-up period was 61.0 months (range: 2.0-149.7). The majority (88.6%) of patients had Stage IA EEC. The 5-year RFS and overall survival rates were 97.5% and 98.5%, respectively. In multivariate analysis for RFS, the significant prognostic factors were lymphovascular invasion (hazard ratio = 4.604; 95% CI: 1.387-15.288; P = 0.013) and grade (grade 2; hazard ratio = 4.949; 95% CI: 1.035-23.654; P = 0.045, and grade 3; hazard ratio = 5.469; 95% CI: 1.435-20.848; P = 0.013). Other pathologic factors including TCC in the fallopian tube lumen, myometrial invasion patterns, PPC, and hormonal status had no prognostic significance. TCC in the fallopian tube lumen, myometrial invasion pattern, PPC, and estrogen and progesterone receptor positivity were not significant prognostic factors in Stage I EEC. In contrast, lymphovascular invasion and grade were significant prognostic factors.

Patient-Derived Tumor Organoids Combined with Function-Associated ScRNA-Seq for Dissecting the Local Immune Response of Lung Cancer.

In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient-derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function-associated single-cell RNA sequencing (FascRNA-seq) platform allowing both phenotypic evaluation and scRNA-seq at single-organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA-seq data of individual pLCOs with their responses to anti-PD-1 (αPD-1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti-tumor immunity, to identify potential tumor-reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA-seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.