High Risk Of Metastasis Research Articles

Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells.

Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer. Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin. Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo. Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.

Positive Association of Serum Vitamin B6 Levels with Intrapulmonary Lymph Node and/or Localized Pleural Metastases in Non-Small Cell Lung Cancer: A Retrospective Study.

The relationship between vitamin B levels and the development and progression of lung cancer remains inconclusive. We aimed to investigate the relationship between B vitamins and intrapulmonary lymph nodes as well as localized pleural metastases in patients with non-small cell lung cancer (NSCLC). This was a retrospective study including patients who underwent lung surgery for suspected NSCLC at our institution from January 2016 to December 2018. Logistic regression models were used to evaluate the associations between serum B vitamin levels and intrapulmonary lymph node and/or localized pleural metastases. Stratified analysis was performed according to different clinical characteristics and tumor types. A total of 1498 patients were included in the analyses. Serum vitamin B6 levels showed a positive association with intrapulmonary metastasis in a multivariate logistic regression (odds ratio (OR) of 1.016, 95% confidence interval (CI) of 1.002-1.031, p = 0.021). After multivariable adjustment, we found a high risk of intrapulmonary metastasis in patients with high serum vitamin B6 levels (fourth quartile (Q4) vs. Q1, OR of 1.676, 95%CI of 1.092 to 2.574, p = 0.018, p for trend of 0.030). Stratified analyses showed that the positive association between serum vitamin B6 and lymph node metastasis appeared to be stronger in females, current smokers, current drinkers, and those with a family history of cancer, squamous cell carcinoma, a tumor of 1-3 cm in diameter, or a solitary tumor. Even though serum vitamin B6 levels were associated with preoperative NSCLC upstaging, B6 did not qualify as a useful biomarker due to weak association and wide confidence intervals. Thus, it would be appropriate to prospectively investigate the relationship between serum vitamin B6 levels and lung cancer further.

Preoperative chemoradiotherapy using tegafur/uracil, oral leucovorin, and irinotecan (TEGAFIRI) followed by oxaliplatin-based chemotherapy as total neoadjuvant therapy for locally advanced rectal cancer: the study protocol for a phase II trial

BackgroundTotal neoadjuvant therapy (TNT) is a novel treatment strategy that is an alternative to preoperative chemoradiotherapy (CRT) for locally advanced rectal cancer (LARC). However, an optimal protocol for TNT has not yet been established. The present study will be an open-label, single-arm, single-center trial to develop a new protocol.MethodsThirty LARC patients at high risk of distant metastasis will receive CRT consisting of long-course radiation, concurrent with tegafur/uracil, oral leucovorin, irinotecan (TEGAFIRI), followed by mFOLFOX-6 or CAPOX before undergoing surgery.DiscussionSince previous findings showed a high percentage of grade 3–4 adverse events with the TEGAFIRI regimen for CRT and TNT, the primary outcome of this study will be safety and feasibility. Our regimen for CRT consists of the biweekly administration of irinotecan for good patient compliance. The novel combination approach of this treatment may improve the long-term outcomes of LARC.Trial RegistrationJapan Registry of Clinical Trials jRCTs031210660.

Prospects for fertility preservation: the ovarian organ function reconstruction techniques for oogenesis, growth and maturation in vitro.

Today, fertility preservation is receiving more attention than ever. Cryopreservation, which preserves ovarian tissue to preserve fertility in young women and reduce the risk of infertility, is currently the most widely practiced. Transplantation, however, is less feasible for women with blood-borne leukemia or cancers with a high risk of ovarian metastasis because of the risk of cancer recurrence. In addition to cryopreservation and re-implantation of embryos, in vitro ovarian organ reconstruction techniques have been considered as an alternative strategy for fertility preservation. In vitro culture of oocytes in vitro Culture, female germ cells induction from pluripotent stem cells (PSC) in vitro, artificial ovary construction, and ovaria-related organoids construction have provided new solutions for fertility preservation, which will therefore maximize the potential for all patients undergoing fertility preservation. In this review, we discussed and thought about the latest ovarian organ function reconstruction techniques in vitro to provide new ideas for future ovarian disease research and fertility preservation of patients with cancer and premature ovarian failure.

KRAS G12C Mutant Non–Small Cell Lung Cancer Linked to Female Sex and High Risk of CNS Metastasis: Population-based Demographics and Survival Data From the National Swedish Lung Cancer Registry

BackgroundReal-world data on demographics related to KRAS mutation subtypes are crucial as targeted drugs against the p.G12C variant have been approved. MethodWe identified 6183 NSCLC patients with reported NGS-based KRAS status in the Swedish national lung cancer registry between 2016 and 2019. Following exclusion of other targetable drivers, three cohorts were studied: KRAS-G12C (n = 848), KRAS-other (n = 1161), and driver negative KRAS-wild-type (wt) (n = 3349). ResultsThe prevalence of KRAS mutations and the p.G12C variant respectively was 38%/16% in adenocarcinoma, 28%/13% in NSCLC-NOS and 6%/2% in squamous cell carcinoma. Women were enriched in the KRAS-G12C (65%) and KRAS-other (59%) cohorts versus KRAS-wt (48%). A high proportion of KRAS-G12C patients in stage IV (28%) presented with CNS metastasis (vs. KRAS-other [19%] and KRAS-wt [18%]). No difference in survival between the mutation cohorts was seen in stage I-IIIA. In stage IV, median overall survival (mOS) from date of diagnosis was shorter for KRAS-G12C and KRAS-other (5.8 months/5.2 months) vs. KRAS wt (6.4 months). Women had better outcome in the stage IV cohorts, except in KRAS-G12C subgroup where mOS was similar between men and women. Notably, CNS metastasis did not impact survival in stage IV KRAS-G12C, but was associated with poorer survival, as expected, in KRAS-other and KRAS-wt. ConclusionThe KRAS p.G12C variant is a prevalent targetable driver in Sweden and significantly associated with female sex and presence of CNS metastasis. We show novel survival effects linked to KRAS p.G12C mutations in these subgroups with implications for clinical practice.

SYTL2 promotes metastasis of prostate cancer cells by enhancing FSCN1-mediated pseudopodia formation and invasion

BackgroundMetastatic prostate cancer (mPCa) has a poor prognosis with limited treatment options. The high mobility of tumor cells is the key driving characteristic of metastasis. However, the mechanism is complex and far from clarified in PCa. Therefore, it is essential to explore the mechanism of metastasis and discover an intrinsic biomarker for mPCa.MethodsTranscriptome sequencing data and clinicopathologic features of PCa from multifarious public databases were used to identify novel metastatic genes in PCa. The PCa tissue cohort containing 102 formalin-fixed paraffin-embedded (FFPE) samples was used to evaluate the clinicopathologic features of synaptotagmin-like 2 (SYTL2) in PCa. The function of SYTL2 was investigated by migration and invasion assays and a 3D migration model in vitro and a popliteal lymph node metastasis model in vivo. We performed coimmunoprecipitation and protein stability assays to clarify the mechanism of SYTL2.ResultsWe discovered a pseudopodia regulator, SYTL2, which correlated with a higher Gleason score, worse prognosis and higher risk of metastasis. Functional experiments revealed that SYTL2 promoted migration, invasion and lymph node metastasis by increasing pseudopodia formation in vitro and in vivo. Furthermore, SYTL2 induced pseudopodia formation by enhancing the stability of fascin actin-bundling protein 1 (FSCN1) by binding and inhibiting the proteasome degradation pathway. Targeting FSCN1 enabled rescue and reversal of the oncogenic effect of SYTL2.ConclusionsOverall, our study established an FSCN1-dependent mechanism by which SYTL2 regulates the mobility of PCa cells. We also found that the SYTL2-FSCN1-pseudopodia axis may serve as a pharmacological and novel target for treating mPCa.

20P Spatial analysis of residual tumor tissue provides new clues for post-neoadjuvant treatment approaches in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a very heterogeneous disease with high metastatic recurrence risk. We showed (PMID 34535679) that TNBCs relapsing rapidly (RR) after NACT (in ≤ 12 months) are highly proliferative, immune-cold, often with large residual cancer burden (RCB) and fatal outcome. No specific biomarkers or therapeutic targets are known for this clinical category. In this study we performed spatial molecular analysis of the RR and the RCB3 TNBCs, with the aim to help improving treatment of the TNBC patients with the poorest prognosis. We analyzed residual tumor tissue samples of TNBC patients treated by standard NACT from 01/01/2009 to 31/12/2021. The cases were classified into RR, standardly-relapsing (SR, 1-5 years post-NACT) and without recurrences (NR). The amount of tumor-infiltrating lymphocytes (TILs) was determined by optical microscopy (H&E). Tissue protein expression was spatially resolved by GeoMx® Digital Spatial Profiler (NanoString Technology). Sixteen, 17 and 47 RR, SR and NR cases were analyzed, respectively. GeoMx analysis included 216 Region-of-Interest (ROIs) of tumor microenvironment (TME) and 207 of tumor cells (TC), with balanced (∼50/50) numbers of peripheral (P) and central (C) ROIs. Tumors and ROIs were classified into TIL-rich (LR) and TIL-poor (LP) using cut-off of 10% TILs. The RR TNBCs showed higher Ki67, cleaved caspase 9, granzyme B (GZMB), FoxP3 and fibronectin (FBNCT) in TME-P-LR ROIs but higher GADPH, TIM-3, PD-L1 and IDO in TME-P-LP ROIs. Compared to TNBC-SR, the RR had higher GZMB and CD34 in TME-P-LR ROIs. In TME-C-LR ROIs of the RRs, CD8 expression was low, as well as the one of CD45, CD3 and CD8 in TME-C-LP. The RCB3 category contained 9, 8 and 10 RR, SR and NR cases, respectively. Although most RCB3 cases were TIL-poor, the NR cases showed significantly higher expression of CD3, CD8, GZMA, CD4, CD56, CD95, LAG3, HLA-DR, BIM and BCL6 than the RR cases. By this study we revealed several potential therapeutic targets (fibronectin, IDO, TIM-3, CD34 /angiogenesis) in the RR TNBCs. Interestingly, we also showed that some RCB3 cases have immune-activated TME and good prognosis. Spatial tissue analysis was crucial for these discoveries.

CD19 (+) B Cell Combined with Prognostic Nutritional Index Predicts the Clinical Outcomes of Patients with Gastric Cancer Who Underwent Surgery.

(1) Background: The aim of this study was to explore the predictive ability of lymphocyte subsets for the prognosis of gastric cancer patients who underwent surgery and the prognostic value of CD19 (+) B cell combined with the Prognostic Nutritional Index (PNI). (2) Methods: This study involved 291 patients with gastric cancer who underwent surgery at our institution between January 2016 and December 2017. All patients had complete clinical data and peripheral lymphocyte subsets. Differences in clinical and pathological characteristics were examined using the Chi-square test or independent sample t-tests. The difference in survival was evaluated using Kaplan-Meier survival curves and the Log-rank test. Cox's regression analysis was performed to identify independent prognostic indicators, and nomograms were used to predict survival probabilities. (3) Results: Patients were categorized into three groups based on their CD19 (+) B cell and PNI levels, with 56 cases in group one, 190 cases in group two, and 45 cases in group three. Patients in group one had a shorter progression-free survival (PFS) (HR = 0.444, p < 0.001) and overall survival (OS) (HR = 0.435, p < 0.001). CD19 (+) B cell-PNI had the highest area under the curve (AUC) compared with other indicators, and it was also identified as an independent prognostic factor. Moreover, CD3 (+) T cell, CD3 (+) CD8 (+) T cell, and CD3 (+) CD16 (+) CD56 (+) NK T cell were all negatively correlated with the prognosis, while CD19 (+) B cell was positively associated with the prognosis. The C-index and 95% confidence interval (CI) of nomograms for PFS and OS were 0.772 (0.752-0.833) and 0.773 (0.752-0.835), respectively. (4) Conclusions: Lymphocyte subsets including CD3 (+) T cell, CD3 (+) CD8 (+) T cell, CD3 (+) CD16 (+) CD56 (+) NK T cell, and CD19 (+) B cell were related to the clinical outcomes of patients with gastric cancer who underwent surgery. Additionally, PNI combined with CD19 (+) B cell had higher prognostic value and could be used to identify patients with a high risk of metastasis and recurrence after surgery.

The Prognostic Value and Clinical Utility of the 40-Gene Expression Profile (40-GEP) Test in Cutaneous Squamous Cell Carcinoma: Systematic Review and Meta-Analysis.

The current tumor staging systems for cutaneous squamous cell carcinoma (cSCC) are considered inadequate and insufficient for evaluating the risk of metastasis and for identifying patients at high risk of cSCC. This meta-analysis aimed to assess the prognostic significance of a 40-gene expression profile (40-GEP) both independently and integrated with clinicopathologic risk factors and established staging systems (American Joint Committee on Cancer, eighth edition (AJCC8) and Brigham and Women's Hospital (BWH)). Electronic databases, including PubMed (MEDLINE), Embase, the Cochrane Library, and Google Scholar, were systematically searched to identify cohort studies and randomized controlled trials on evaluations of the prediction value of 40-GEP in cSCC patients up to January 2023. The metastatic risk analysis of a given 40-GEP class combined with tumor stage and/or other clinicopathologic risk factors was based upon log hazard ratios (HRs) and their standard error (SE). Heterogeneity and subgroup analyses were performed, and data quality was assessed. A total of 1019 patients from three cohort studies were included in this meta-analysis. The overall three-year metastatic-free survival rates were 92.4%, 78.9%, and 45.4% for class 1 (low risk), class 2A (Intermediate risk), and class 2B (high risk) 40-GEP, respectively, indicating a significant variation in survival rates between the risk classification groups. The pooled positive predictive value was significantly higher in class 2B when compared to AJCC8 or BWH. The subgroup analyses demonstrated significant superiority of integrating 40-GEP with clinicopathologic risk factors or AJCC8/BWH, especially for class 2B patients. The integration of 40-GEP with staging systems can improve the identification of cSCC patients at high risk of metastasis, potentially leading to improved care and outcomes, especially in the high-risk class 2B group.

Regional lymphadenectomy in the treatment of patients with melanoma skin with metastases in the sentinel lymph nodes

Regional recurrences are one of the most common first manifestations of progression of clinically localized cutaneous melanoma. They are caused by subclinical metastases in the lymph nodes. Biopsy of the sentinel lymph nodes is the most effective method of detection of hidden regional metastases. It allows to appropriately increase the volume of surgical treatment prior to dissection of the affected lymph collector and perform adjuvant therapy with the most effective drugs. This, in its turn, allows for significant improvement of regional and systemic control of the disease and long-term treatment outcomes.Data obtained in several randomized trials on the effectiveness of lymph node dissection in patients with sentinel lymph node metastasis showed that this procedure significantly improves regional control of the disease but does not affect long-term treatment outcomes. Additionally, some studies showed that assignment of responsibility for regional control to adjuvant therapy does not meet the expectations. Therefore, the most effective method of regional control of cutaneous melanoma is surgery. All of these studies show that it is necessary to develop criteria for identification of patients with high risk of metastases in the non-sentinel lymph nodes who require lymph node dissection for regional disease control and creation of the best conditions for adjuvant systemic therapy. This requires identification of predictors of multiple involvement of regional lymph nodes justifying indications for radical lymph node dissection. Studies in this area are being performed but their results are ambiguous and sometimes contradictory.Aim. To evaluate the effectiveness of lymph node biopsy and radical lymph node dissection in patients with cutaneous melanoma and subclinical involvement of the sentinel lymph node.

Cerium End-Deposited Gold Nanorods-Based Photoimmunotherapy for Boosting Tumor Immunogenicity.

Triple-negative breast cancer (TNBC) was closely related to high metastatic risk and mortality and has not yet found a targeted receptor for targeted therapy. Cancer immunotherapy, especially photoimmunotherapy, shows promising potential in TNBC treatment because of great spatiotemporal controllability and non-trauma. However, the therapeutic effectiveness was limited by insufficient tumor antigen generation and the immunosuppressive microenvironment. We report on the design of cerium oxide (CeO2) end-deposited gold nanorods (CEG) to achieve excellent near-infrared photoimmunotherapy. CEG was synthesized through hydrolyzing of ceria precursor (cerium acetate, Ce(AC)3) on the surface of Au nanorods (NRs) for cancer therapy. The therapeutic response was first verified in murine mammary carcinoma (4T1) cells and then monitored by analysis of the anti-tumor effect in xenograft mouse models. Under near-infrared (NIR) light irradiation, CEG can efficiently generate hot electrons and avoid hot-electron recombination to release heat and form reactive oxygen species (ROS), triggering immunogenic cell death (ICD) and activating part of the immune response. Simultaneously, combining with PD-1 antibody could further enhance cytotoxic T lymphocyte infiltration. Compared with CBG NRs, CEG NRs showed strong photothermal and photodynamic effects to destroy tumors and activate a part of the immune response. Combining with PD-1 antibody could reverse the immunosuppressive microenvironment and thoroughly activate the immune response. This platform demonstrates the superiority of combination therapy of photoimmunotherapy and PD-1 blockade in TNBC therapy.

Theranostics of Primary Prostate Cancer: Beyond PSMA and GRP-R.

First, we prospectively performed neurotensin receptor-1 (NTS1) IHC in a series of patients receiving both [68Ga]Ga-PSMA-617 and [68Ga]Ga-RM2 before prostatectomy. In this series, PSMA and GRP-R IHC were also available (n = 16). Next, we aimed at confirming the PSMA/GRP-R/NTS1 expression profile by retrospective autoradiography (n = 46) using a specific radiopharmaceuticals study and also aimed to decipher the expression of less-investigated targets such as NTS2, SST2 and CXCR4. In the IHC study, all samples with negative PSMA staining (two patients with ISUP 2 and one with ISUP 3) were strongly positive for NTS1 staining. No samples were negative for all three stains-for PSMA, GRP-R or NTS1. In the autoradiography study, binding of [111In]In-PSMA-617 was high in all ISUP groups. However, some samples did not bind or bound weakly to [111In]In-PSMA-617 (9%). In these cases, binding of [111n]In-JMV 6659 and [111In]In-JMV 7488 towards NTS1 and NTS2 was high. Targeting PSMA and NTS1/NTS2 could allow for the detection of all intraprostatic lesions.

Integrative analyses of ferroptosis and immune related biomarkers and the osteosarcoma associated mechanisms

Osteosarcoma (OS) is the most common primary malignant bone tumor with high metastatic potential and relapse risk. To study the regulatory mechanism of the OS microenvironment, a complex regulatory network involving the ferroptosis- and immune response-related genes remains to be established. In the present study, we determined the effect of a comprehensive evaluation system established on the basis of ferroptosis- and immune-related genes on the immune status, related biomarkers, prognosis, and the potential regulatory networks underlying OS based on the TARGET and Gene Expression Omnibus databases that contain information on OS patients by bioinformatics analyses. We first characterized individual ferroptosis scores and immune scores through gene set variation analysis (GSVA) against TARGET-OS datasets. We then identified differentially expressed genes by score groups. Weighted gene co-expression network analysis was performed to identify the most relevant ferroptosis-related and immune-related gene modules, which facilitated the identification of 327 ferroptosis gene and 306 immune gene candidates. A 4-gene (WAS, CORT, WNT16, and GLB1L2) signature was constructed and valuation using the least absolute shrinkage and selection operator-Cox regression models to effectively predict OS prognosis. The prediction efficiency was further validated by GSE39055. We stratified patients based on the prognostic scoring systems. Eight hub genes (namely CD3D, CD8A, CD3E, IL2, CD2, MYH6, MYH7, and MYL2) were identified, and TF–miRNA target regulatory networks were constructed. Furthermore, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, gene set enrichment analysis, and GSVA were used to determine the signature’s potential pathways and biological functions, which showed that the hub genes were enriched in ferroptosis-associated biological functions and immune-associated molecular mechanisms. Thereafter, we investigated the proportion and infiltration extent of 22 infiltrating immune cells by using CIBERSORT, which revealed significant subgroup differences in CD8 + T cells, M0 macrophages, and M2 macrophages. In conclusion, we determined a new ferroptosis-related and immune-related gene signature for predicting OS patients’ prognosis and further explored the ferroptosis and immunity interactions during OS development, which provides insights into the exploration of molecular mechanisms and targeted therapies in patients with OS.

Diagnostic Aqueous Humor Proteome Predicts Metastatic Potential in Uveal Melanoma.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.

Primary signet ring cell carcinoma of the breast: A case report and literature review

ABSTRACT Primary signet ring cell carcinoma (SRCC) of the breast is a rare and aggressive type of breast cancer characterized by increased intracellular mucin production. It has a high risk of metastasis and poor prognosis compared to other breast cancer types. We report a 56-year-old woman with primary SRCC of the breast who first presented with retraction on her left breast. Radiological examination revealed a mass that causes the retraction. The patient underwent left modified radical mastectomy, and pathology results showed a 70% signet ring cell pattern. Chemotherapy consists of an adriamycin-cyclophosphamide regimen administered. in this case, we aim to review the literature on this topic and inform the physicians.

Abstract 3940: Validation of TFX1, a first-in-class target for cancer metastasis

Abstract Most of the drugs approved for metastatic epithelial carcinomas treat the proliferation of the tumor and not the biology of metastasis. To date, all clinical trials around metastasis that focused on proteases, kinases, or integrins have failed in clinics. As a result, metastasis biology-focused drug discovery has been deprioritized.We asked the question as to what the rate-limiting steps of metastasis were. To answer it, we created a cell-based functional assay platform, METAssay™, that dissected all metastasis biology steps into thirty cellular assays and characterization steps. The platform identified the functional differences between metastatic and proliferating cells. This platform was normalized with patient tumor cells, and a machine learning algorithm, METSCAN™, was subsequently applied to identify the weighted steps that contributed towards successful metastasis. This led to the identification of four first-in-class targets; two transcription factors, one cytokine receptor and one nuclear hormone receptor.We then validated the relevance of the first target, TFX1, by overexpressing it in both colorectal and triple-negative breast cancer cell lines. Interestingly, apart from the expected increase in migration and invasion, all cell lines showed an increase in ROS, Autophagy, PDL-1, CD-73, quiescence, exosome uptake and a decrease in apoptosis, mitosis, and exosome secretion. Conversely, the downregulation of TFX1 showed the opposite effect. TFX1 was purified, and a biochemical isothermal calorimetry (ITC) assay was standardized with a tool compound with a KD of 107 nM. The compound effectively reduced metastatic properties in the cell lines and in multiple patient samples, as analyzed by the METAssay™ platform. Colorectal cancer cell lines overexpressing TFX1 also showed increased platelet binding, which was reduced by the tool compound, but not triple-negative cancer cell lines. Also, the effect of overexpression in non-metastatic cells was more profound than in metastatic cells, suggesting a saturating effect. Further characterization of the tool compound is currently ongoing to help establish a baseline for a novel discovery programme.The ideal drug to delay metastasis should be given to primary tumor patients with no evidence of clinical metastasis, irrespective of their pathological grading. Therefore, it should have a high bar for safety. Our goal is to collaborate on a discovery journey to identify candidates and create a companion diagnostic that will identify primary tumor patients at high risk of metastasis, thereby selecting the right patient cohort for clinical trials. Citation Format: Arnab Roy Chowdhury, Manoj Pandre, Chinmaya Amarkanth, Debabani Roy Chowdhury, John Ellingboe. Validation of TFX1, a first-in-class target for cancer metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3940.

Abstract 5553: Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma

Abstract Introduction: Approximately half of patients with uveal melanoma (UM), a primary eye cancer, will develop metastatic disease. Gene expression profiling (GEP) is clinically validated to stratify risk of metastasis by assigning UM patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy which are limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding or tumor dissemination. Thus, liquid biopsy has emerged as a significantly less invasive alternative. Blood biopsy has largely been unsuccessful for clinical use in UM due to low detection rates in the setting disease limited to the eye, however eye-specific aqueous humor (AH) liquid biopsy may be a better alternative. In this study, we seek to determine the AH proteome related to the advanced GEP class 2 using the diagnostic AH specimens. Method: Twenty UM treatment naive AH were collected before plaque brachytherapy. Patients were sub-grouped by GEP classes into GEP 1 (n=12) and GEP 2 (n=5). Three patients were classified as GEP unknown (GEP NA, n=3) due to the unavailability of tumor biopsy. Ten microliters of AH samples were analyzed by proximity extension assay-derived multiplexed Olink platform. Protein expression levels of 1472 targets were analyzed, compared between GEP classes and correlated with clinical features. Significant differentially expressed proteins (DEPs, fold-change (FC) &amp;gt; 2 or FC &amp;lt; 0.5, P &amp;lt; 0.01) were subjected to Qiagen ingenuity pathway analysis for cellular pathway and upstream regulator identification. Results: GEP 2 class was correlated with AJCC stages (P = 0.012), advanced clinical tumor stages (P = 0.007) and mutated BAP1 (P = 0.018). 45 DEPs were identified when comparing GEP classes. Among them, 31 are up-regulated DEPs [fold-change (FC) &amp;gt;2, P &amp;lt; 0.01] and 14 are down-regulated DEPs (FC &amp;lt; 0.5, P &amp;lt; 0.01) in GEP 2 compared to GEP1. The unsupervised clustering analysis showed that the 45 DEPs well-differentiate AH samples by GEP classes, and the 3 GEP NA samples were clustered with GEP1 class. Pathway analysis showed that 45 DEPs contribute to metastatic-related pathways including cellular movement, cellular proliferation and epithelial to mesenchymal transition. Two upstream regulators, IL1 receptor and SPRY2, were predicted to regulate 8 out of the 45 DEPs. IL1R2 (FC = 3.4, P = 0.021) and SPRY2 (FC = 0.6, P = 0.052) protein expression levels were found matched as prediction in our Olink dataset. Conclusions: 45 AH DEPs could differentiate GEP class 1 and 2 at the diagnostic stage and could be detected even when the tumor was too small to biopsy. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy. Citation Format: Chen-Ching Peng, Liya Xu, Jesse Berry. Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5553.

Abstract 1306: New therapeutic approach for triple negative breast cancer: soluble TNFα blockade and MUC4 expression as a prognostic biomarker

Abstract Among all breast cancers, triple negative (TNBC) subtype has the worst survival rate. We have demonstrated that the proinflammatory cytokine TNFα induces trastuzumab resistance through Mucin4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein expressed in several tumors and it is involved in metastasis dissemination. Here, we evaluated the role of TNFα and MUC4 in metastasis in TNBC cell lines and we assessed the clinical impact of MUC4 expression in TNBC patients. TNFα blockade was achieved using etanercept (E), which blocks the soluble (sTNFα) and transmembrane isoforms of TNFα, or dominant negative protein INB03 (DN) which only neutralizes sTNFα. BT-549 and MDA-MB-231 TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. TNFα blockade decreases the expression of the mesenchymal markers vimentin and snail in both cell lines. To assess the impact of TNFα blockade on tumor cells and its effect on the tumor microenvironment (TME), we collected conditioned media (CM) of MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 was impaired with CM-Eta (p&amp;lt;0.01) and CM-DN (p&amp;lt;0.01), and MDA-MB-231 invasive capacity was reduced only with CM-DN (p&amp;lt;0.01). We evaluated MUC4 participation on invasion in MDA-MB-231 cell line transfected with a siRNA targeting MUC4 with or without DN treatment, and an invasion assay was performed. MUC4 knockdown impaired cell invasion (p=0.0001 vs siRNA control transfected cells). The treatment with DN of the MUC4-silenced cells did not further enhanced the effect, demonstrating that the impairment of the invasion is due to the decrease in MUC4. We determined the presence of TILs by H&amp;E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 55 TNBC patients. MUC4 expression inversely correlated with TILs (p=0.00013). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM on T cell migration. CM-DN increased T cell migration compared to control CM (p&amp;lt;0.01). MUC4 expression inversely correlated with Ki67 (p=0.016), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher metastasis risk (p=0.005).In conclusion, TNFα blocking agents decrease MUC4, mesenchymal markers and the invasive capacity of TNBC cells, and in turn increase human T cell migration. MUC4 is an independent biomarker of poor overall survival, it is associated with an increased risk of metastasis and immune desert tumors. We propose TNFα as a new target for the treatment of TNBC, and MUC4 as a predictive marker to guide a combined treatment of TNFα blockers with chemotherapy or immunotherapy. Citation Format: Florencia Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, María Florencia Mercogliano, Roxana Schillaci. New therapeutic approach for triple negative breast cancer: soluble TNFα blockade and MUC4 expression as a prognostic biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1306.

Primary angiosarcoma of the breast: a case report

Background: Angiosarcoma of the breast is a rare form of breast malignancy. Primary breast angiosarcoma occurs sporadically primarily in young women while secondary breast angiosarcoma most frequently develops in older patients with prior history of radiation therapy or chronic lymphedema. This aggressive malignant tumor of the vascular endothelium has a high risk of local recurrence and distant metastasis and the diagnosis carries a poor prognosis.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities.

Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC-TCGA-UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour-associated macrophages and hypoxia genes, suggesting PLIN2-dependent changes in oxygen and lipid metabolism in the TME of low and high-metastatic risk uveal melanoma. We designed four adipophilin-based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival-functional network approach. Adipophilin-based multigene signatures were validated in BAP1-positive and BAP1-negative uveal melanoma cell lines using a next-generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co-cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.