Lymph Node Research Articles

Overestimation of clinical N-staging in microsatellite instable gastric cancers is associated with VEGF-C signaling and CD8+ T-cell dynamics

Abstract Background Microsatellite instable (MSI) gastric cancers exhibit reduced lymph node (LN) metastasis and improved survival compared to microsatellite stable (MSS) counterparts. However, to our longstanding observation, clinical N-staging (cN) is frequently overestimated in MSI cases. The clinical implications and underlying mechanisms of this discrepancy warrant further investigation. Materials and methods We conducted a comprehensive review of clinicopathological data from a 141 MSI and 1119 MSS gastric cancer patients. Expression of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 were assessed using qPCR and immunohistochemistry. High-parameter flow cytometry was employed to analyze subsets of CD8+ T cells within the tumors. Results Multivariate analysis revealed that MSI status was an independent prognostic factor, alongside the LN ratio and AJCC8 pathology staging. MSI gastric cancers exhibited a reduced LN ratio, particularly at advanced T-staging, compared to MSS counterparts, while maintaining an equivalent LN yield. Overestimation of cN by computed tomography preoperatively was frequent in MSI gastric cancers but was more commonly underestimated in MSS counterparts. VEGF-C and VEGFR-3 expression were lower in MSI tumors. MSI gastric cancers showed an increased total number of CD8+ T cells, albeit with a lower proportion of effector memory cells expressing CD45RA (EMRA) and CD8+ CXCR4+ T cells, compared to MSS counterparts. Conclusion Frequent overestimation of clinical N-staging in MSI gastric cancers is associated with VEGF-C signaling and CD8+ T-cell dynamics and should be cautiously interpreted, as it might misguide therapeutic options.

The Impact of Balloon Use During Endobronchial Ultrasound: A Randomized Pilot Study

Introduction During endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA), a saline-filled balloon placed over the tip of the bronchoscope is used to improve coupling of the ultrasound transducer to the airway wall. However, it is unknown whether it objectively improves image quality or affects procedural outcomes. Our aim is to establish whether the use of a balloon during EBUS impacts image quality, diagnostic yield, procedure duration or complications. Methods A pilot randomized control trial of patients undergoing EBUS-TBNA of mediastinal lymph nodes was performed at a single academic centre. Patients were randomized to use of a saline-filled balloon (versus no balloon use) during EBUS-TBNA of the right lower paratracheal lymph node (station 4R). The EBUS videos were recorded and scored on a 4-point Likert scale by three blinded external reviewers. The primary outcome of this study was ultrasound image quality. Secondary outcomes included diagnostic yield, procedure duration and complications. Results Forty-six patients were randomized. In the balloon group, 61% of patients had an image quality score of “excellent” or “good,” compared to 47% in the no balloon group (p = 0.009). There was no significant difference in diagnostic yield, procedure duration or complications between the groups. Conclusion Inflation of the balloon during EBUS-TBNA at the right lower paratracheal lymph node improves ultrasound image quality but does not impact diagnostic yield, procedure duration or safety. This study provides a framework for additional studies with complete nodal assessment to determine if balloon use has a clinically meaningful benefit on procedural outcomes.

Breaching the Barrier: Investigating Initial Herpes Simplex Viral Infection and Spread in Human Skin and Mucosa

Herpes simplex virus (HSV) is sexually transmitted via the anogenital mucosa where it initially infects epidermal keratinocytes and mononuclear phagocytes (MNPs). It then spreads to the dorsal root ganglion via sensory nerve endings, to remain latent for life with periodic reactivation. Currently, there is no cure or vaccine. Initial or recurrent HSV infection can produce serious complications and mediate acquisition of HIV. This review outlines the initial events after the HSV infection of human anogenital mucosa to determine the optimal window to target the virus before it becomes latent. After infection, HSV spreads rapidly within the mid-layers of epidermal keratinocytes in the explanted human inner foreskin. Infected cells produce chemokines, which modulate nectin-1 distribution on the surface of adjacent keratinocytes, facilitating viral spread. Epidermal Langerhans cells and dendritic cells become infected with HSV followed by a “viral relay” to dermal MNPs, which then present viral antigen to T cells in the dermis or lymph nodes. These data indicate the need for interruption of spread within 24 h by diffusible vaccine-induced mediators such as antiviral cytokines from resident immune cells or antibodies. Intradermal/mucosal vaccines would need to target the relevant dermal MNPs to induce HSV-specific CD4+ and CD8+ T cells.

In a prospective, multicenter study, the 31-GEP identified patients at increased risk of tumor recurrence and added significant prognostic value to AJCC staging

Cutaneous melanoma (CM) guidelines base management decisions on a patient’s American Joint Committee on Cancer (AJCC) tumor stage. However, limitations in staging accuracy suggest additional tools could improve risk-aligned patient management decisions. The 31-gene expression profile (GEP) test identifies patients with CM with low (Class 1A), intermediate (Class 1B/2A), or high (Class 2B) risk for sentinel lymph node (SLN) positivity, recurrence, metastasis, and death. In this multicenter, prospective study, we validated the 31-GEP for risk of recurrence and demonstrated the added value of 31-GEP to AJCC staging. Patients were included in the prospective CONNECTION study if they were tested with the 31-GEP from 2018 to the present (n=876). Survival was estimated using Kaplan-Meier analysis and 31-GEP stratification tested with the log-rank test. Cox regression was performed to identify predictors of recurrence. ANOVA was used to compare Cox models for the most accurate recurrence prediction. Patients with a Class 1A result had significantly higher 3-year recurrence-free survival than those with a Class 1B/2A or Class 2B result (98.5% vs. 80.8% vs. 63.0%, p<0.001). The 31-GEP had higher sensitivity (75.0% vs. 37.5%) and negative predictive value (98.6% vs. 96.9%) than AJCC staging. Multivariable analysis demonstrated that Class 1B/2A (hazard ratio, HR=3.12, p=0.037) and Class 2B (HR=5.91, p=0.002) were significant predictors of recurrence in addition to stage IB (HR=4.02, p=0.02), IIA (HR=6.06, p=0.006), IIC (HR=17.14, p<0.001), and III (HR=8.84, p<0.001). Stage IIB (HR=4.63, p=0.057) was not significant. Comparing a staging-only model to a 31-GEP+staging model showed that adding 31-GEP to staging significantly improved prediction accuracy over staging alone (ANOVA: c2=10.58, p=0.005). In this prospective study, the 31-GEP stratified risk of recurrence, was a significant predictor of recurrence, and added significant predictive value to AJCC staging. The 31-GEP identifies patients at high risk of recurrence who should be managed more intensely, and adding 31-GEP to staging allows better risk-aligned care decisions, which can lead to improved patient outcomes.

In-depth analysis of lymph node metastasis-related sialylated protein profiling and their clinical and biological significance in colorectal cancer using mass spectrometry and multi-omics technologies

In-depth analysis of lymph node metastasis-related sialylated protein profiling and their clinical and biological significance in colorectal cancer using mass spectrometry and multi-omics technologies

ASO Author Reflections: Log Odds of Metastatic Lymph Nodes After Curative-Intent Resection of Gallbladder Cancer

ASO Author Reflections: Log Odds of Metastatic Lymph Nodes After Curative-Intent Resection of Gallbladder Cancer

Predicting pan-cancer immune-checkpoint therapy and prognosis with a chromatin-accessibility-related alternative splicing signature: a retrospective study

Background: This study examines alternative splicing (AS) events in genes linked to chromatin accessibility in various cancers and their relation to the tumor immune microenvironment. Methods: Data from the Cancer Genome Atlas Database (TCGA) were used to identify independent prognostic factors for pan-cancer. We explored the correlation between differentially expressed genes and tumor immunity, including immune checkpoint genes, tumor development, and immune cells. A regulatory network diagram of alternative splicing-splicing factors (AS-SFs) was constructed to find potential immunotherapy targets. Results: IRF5 and E2F8 genes showed significant differential expression in pan-cancer. Age, cancer grade, primary tumor, cancer lymph nodes, and distant metastasis were independent prognostic factors. The risk model achieved good predictive performance, with AUC values of 0.705, 0.746, 0.743, and 0.743 for 1-year, 3-year, 5-year, and 10-year survival predictions, respectively. Positive correlations were found between IRF5/E2F8 and CD274/CTLA4 in certain cancers using TIMER and CIBERSORT software. Conclusions: AS events in chromatin accessibility genes (IRF5 and E2F8) have significant predictive value in pan-cancer prognosis. Our model assesses patient survival probability and highlights the synergistic impact of immune checkpoints and the AS-SF regulatory network on tumor immunotherapy.

Letter to the Editor Regarding: “Lymphedema Rates Following Axillary Lymph Node Dissection with and Without Immediate Lymphatic Reconstruction: A Prospective Trial” by Jakub et al.

Letter to the Editor Regarding: “Lymphedema Rates Following Axillary Lymph Node Dissection with and Without Immediate Lymphatic Reconstruction: A Prospective Trial” by Jakub et al.

Clinical and Sonographic Differences between RET Fusion-Positive and BRAFV600E in Papillary Thyroid Carcinoma

Abstract Purpose This study aimed to describe the ultrasound characteristics of papillary thyroid carcinoma (PTC) harboring RET gene fusion and explore its clinical significance. Methods A retrospective study was conducted on 209 patients with PTC diagnosed between Aug 2021 and Jan 2024. All patients underwent ultrasound examination and were confirmed to be positive for RET fusion or BRAFV600E by pathological results. The differences in clinical characteristics and ultrasonography features between the two groups were analyzed. Results Among all PTCs (n=209), we detected 30 RET fusions, and 179 BRAFV600E. RET-fusion PTCs showed younger age (38.5(16.0-69.0) vs. 42.9(20.0-74.0) years, p<.05), larger tumor size (1.09(0.5-4.0) vs. 0.77(0.1-4.0) cm, p<.005), and more advanced N stage (p<.001) than BRAFV600E PTCs. RET-fusion PTCs were mainly classical and diffuse sclerosing subtypes. In terms of ultrasound performance, RET-fusion PTCs were mainly manifested as heterogeneous echogenicity (43.3%), ill-defined tumor margin (90.0%), irregular shape (83.3%) and intranodular microcalcification (83.3%), characterized by scattered microcalcification around the tumor/within thyroid gland (40.0%). In comparison, BRAFV600E PTCs were mainly characterized by hypoechogenicity (95.5%), round/oval shape (80.4%), and intranodular non-calcification (54.2%). Multivariate logistic regression analysis revealed that scattered microcalcification around the tumor/within the normal gland was an independent risk factor for lateral lymph node metastasis (LLNM) in RET-fusion PTCs (OR 9.79, 95% CI, 1.31, 72.93, P=.026). Conclusions Patients diagnosed with PTC harboring RET fusion presented with distinctive clinical characteristics and sonographic patterns, underscoring the unique diagnostic value of ultrasound examination. It can provide a preoperative non-invasive primary screening method for RET-fusion diagnosis, thus facilitating targeted patients with purposeful molecular sequencing to improve treatment outcomes.

Literature Review on Stages of Colorectal Cancer

Colorectal cancer, primarily originating from polyps in the colon or rectum, represents a significant health challenge due to its potential progression to malignancy. Polyps, abnormal tissue growths in the inner lining of the colon or rectum, often start as benign adenomas. Over time, these adenomas may undergo cellular mutations, transforming into cancerous tumors. Early detection and removal of polyps during screening significantly reduce the likelihood of cancer development. If left untreated, colorectal cancer can invade deeper layers of the colon wall, metastasize to nearby lymph nodes, and spread to distant organs, commonly the liver, peritoneum, or lungs. Colon cancer typically originates in the mucosa, the innermost lining of the colon, and progresses through the tissue and muscle layers over a span of approximately 10 years. The anatomy of the colon and rectum, consisting of the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum, plays a critical role in understanding the disease's progression. Advances in screening methods and treatments have significantly improved outcomes, emphasizing the importance of early intervention in reducing colorectal cancer mortality.

NEDD4L contributes to ferroptosis and cell growth inhibition in esophageal squamous cell carcinoma by facilitating xCT ubiquitination

AbstractThe oncogene xCT plays an indispensable role in tumor growth by protecting cancer cells from oxidative stress and ferroptosis. Emerging evidence indicated xCT function is tightly controlled by posttranslational modifications, especially ubiquitination. However, it still remains unclear what specific regulatory mechanism of xCT by ubiquitin ligases in human cancers. Here, we reported that NEDD4L, an E3 ubiquitin ligases, inhibited esophageal squamous cell carcinoma (ESCC) tumor growth and facilitated ferroptosis by ubiquitination of xCT. NEDD4L expression was declined in ESCC and was associated with tumor invasion, lymph node metastasis and distant metastasis. Silencing NEDD4L triggered ESCC tumor growth. Meanwhile, knock down of NEDD4L prevented the accumulation of ROS, elevated the level of GSH, reduced the content of MDA in ESCC cells, thereby inhibiting ferroptosis. Mechanistically, NEDD4L directly bound to the ∆CT domain of xCT through its WW and HECT domain. More importantly, NEDD4L promoted xCT degradation by facilitating its polyubiquitination in ESCC cells. Collectively, these findings suggest that NEDD4L is crucial in governing the stability of xCT and mediating ferroptosis in ESCC.

Features of Lateral Pelvic Lymph Nodes Associated With Pathological Involvement After Total Neoadjuvant Therapy in Patients Undergoing Lateral Pelvic Lymph Node Dissection

BACKGROUND: There is a lack of consensus regarding treating involved lateral pelvic lymph nodes in rectal cancer. OBJECTIVE: This study aimed to evaluate the clinical and magnetic resonance imaging-based factors associated with pathological lateral pelvic lymph node metastasis in patients undergoing total neoadjuvant therapy and lateral pelvic lymph node dissection. DESIGN: This is a retrospective study. SETTINGS: This study was conducted at a single comprehensive cancer center. PATIENTS: A total of 107 patients with advanced low rectal cancer with pretreatment enlarged lateral pelvic lymph nodes (≥7 mm in long-axis) undergoing total neoadjuvant therapy with long-course chemoradiotherapy, followed by total mesorectal excision and lateral pelvic lymph node dissection, were enrolled. MAIN OUTCOME MEASURES: Pathological lateral pelvic lymph node metastasis and survival. RESULTS: Among 107 patients, 48 had a short-axis lateral node diameter <7 mm at baseline, while 59 had ≥7 mm. The ≥7 mm group showed significantly higher rates of pathological lateral pelvic lymph node metastasis (44.1% vs. 2.1%; p < 0.0001). In this group, pathological lateral pelvic lymph node metastasis was independently associated with pre-treatment malignant features and post-treatment short-axis diameter ≥4 mm. Five-year relapse-free survival was significantly lower in patients with post-treatment lateral node diameter ≥ 4 mm than those with <4 mm (71.1% vs. 86.2%, p = 0.0364). Patients with pathological lateral pelvic lymph node metastasis had significantly lower overall survival, relapse-free survival, and local recurrence-free survival rates. LIMITATIONS: Selection bias exists in a retrospective analysis. CONCLUSIONS: Pathological lateral pelvic lymph node metastasis is rare in patients with pre-treatment short-axis diameter <7 mm. In patients with pre-treatment short-axis diameter ≥7 mm, pre-treatment malignant features and post-treatment short-axis diameter are both associated with pathological lateral pelvic lymph node metastasis. These factors should be considered when deciding whether to proceed with lateral pelvic lymph node dissection after total neoadjuvant therapy. See Video Abstract.

Corrigendum: Predictive factor of large‐volume central lymph node metastasis in clinical N0 papillary thyroid carcinoma patients underwent total thyroidectomy

Corrigendum: Predictive factor of large‐volume central lymph node metastasis in clinical N0 papillary thyroid carcinoma patients underwent total thyroidectomy

ASO Author Reflections: The Minimum Number of Examined Lymph Nodes in Thyroidectomy

ASO Author Reflections: The Minimum Number of Examined Lymph Nodes in Thyroidectomy

Predictors of Recurrence After Curative Surgery for Stage I Colon Cancer: Retrospective Cohort Analysis of the Italian Society of Surgical Oncology Colorectal Cancer Network Collaborative Group

Objective: The aim of this study is to provide solid evidence to update the management of stage I colon cancer (CC) after surgery. Background: Given the low risk of recurrence of stage I CC, some international guidelines do not recommend intensive follow-up after surgery. However, data on the actual incidence, risk factors, and site of recurrences are scarce. Methods: This is a retrospective multicenter cohort study considering patients who underwent surgery at 25 Italian centers between 2010 and 2019, with a minimum follow-up of 24 months. A total of 1883 consecutive adult patients with stage I CC treated with curative surgery were considered, and 1611 fulfilled the inclusion criteria. The primary outcome was the rate of recurrence. Secondary outcomes included survival and risk factors for recurrence. Results: Eighty patients developed cancer recurrence (5.0%), of which 90% was systemic relapse. The event was more frequent in pT2 (6.0% vs 3.2%, P = 0.013), male patients (6.1% vs 3.6%, P = 0.021), in the presence of lymphovascular invasion (7.2% vs 3.6%, P = 0.01), and in cases of partial resection (11.1% vs 4.6%, P = 0.011). Also, preoperative carcinoembryonic antigen (P = 0.007) and tumor diameter (P < 0.001) were higher in the group who relapsed. Most patients had isolated cancer recurrence (90%). Recurrences peaked between 10 and 18 months after surgery and declined over time. Adjusted Cox regression analysis identified tumor diameter, carcinoembryonic antigen level, lymphovascular invasion, male gender, and less than 12 analyzed lymph nodes as significant risk factors for worse recurrence-free survival. Conclusions: This study showed that a not negligible rate of stage I CC recur after curative surgery. Most relapses occur at a single site within the first 3 years after surgery. This evidence could be used to optimize postoperative follow-up.

IL-2/anti-IL-2 antibody complexes augment immune responses to therapeutic cancer vaccines

One driver of the high failure rates of clinical trials for therapeutic cancer vaccines is likely the inability to sufficiently engage conventional dendritic cells (cDCs), the antigen-presenting cell (APC) subset that is specialized in priming antitumor T cells. Here, we demonstrate that, relative to vaccination with an injectable mesoporous silica rod (MPS) vaccine alone (Vax), combining MPS vaccines with CD122-biased IL-2/anti-IL-2 antibody complexes (IL-2cx) drives ~3-fold expansion of cDCs at the vaccination sites, vaccine-draining lymph nodes, and spleens of treated mice. Furthermore, relative to Vax alone, Vax+IL-2cx led to a ~3-fold increase in the numbers of CD8 + T cells and ~15-fold increase in the numbers of NK cells at the vaccination site. Notably, with both the model protein antigen OVA as well as various peptide neoantigens, Vax+IL-2cx induced ~5 to 30-fold greater numbers of circulating antigen-specific CD8 + T cells relative to Vax alone. We further demonstrate that Vax+IL-2cx leads to significantly improved efficacy in the MC38 colon carcinoma model relative to either monotherapy alone, driving complete regressions in 50% of mice in a cDC-dependent manner. Relative to vaccine alone, Vax+IL-2cx led to comparable numbers of CD8 + T cells, but markedly greater numbers of NK cells and activated cDCs in the B16F10 melanoma tumor microenvironment post-therapy. Taken together, these findings suggest that the administration of factors that engage both the cDC-CD8 + T cell and cDC-NK cell axes can boost the potency of therapeutic cancer vaccines.

Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis

Serum small extracellular vesicles-derived BST2 as a biomarker for papillary thyroid microcarcinoma promotes lymph node metastasis

Abstract PR002: Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis

Abstract The majority of cancer-associated deaths result from distant organ metastasis, yet the mechanisms that enable this process remain poorly understood. Colonization of locoregional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it has been unclear whether LN metastasis plays a functional role in disease progression. LNs are major sites of anti-tumor lymphocyte education, including in the context of immunotherapy, yet LN metastasis frequently correlates with further disease progression. By leveraging our mouse models of LN metastasis, we find that LN colonization represents a critical step in tumor progression by inducing tumor-specific immune tolerance in a manner that promotes further dissemination of tumors to distant organs. This promotion of distant seeding by LN metastases is reliant upon adaptive immunity and is tumor specific. Using flow cytometry and single-cell sequencing to perform comprehensive immune profiling, we identify multiple cellular mediators of tolerance. LN metastases evade NK cells, induce shifts in DC and B cell populations, and promote CD8 T cell exhaustion, but of particular importance, we find that regulatory T cells (Tregs) are enriched in the LNs of mice and patients with LN metastases. LN metastatic cells exhibit an enhanced capacity to induce Treg differentiation from naïve CD4 T cells, and ablation of Tregs using FoxP3-DTR mice eliminates the ability of LN metastases to promote distant metastasis. Furthermore, adoptive transfer of Tregs from the LNs of mice bearing LN metastases to naïve mice facilitates lung metastasis in a manner that Tregs from mice without LN metastases cannot. These Tregs are induced in a tumor antigen-specific manner and TCR recognition of tumors is required for the metastasis-promoting tolerance. Using photoconvertible mice, we track the trafficking dynamics of Tregs from tumor involved LNs and demonstrate their elevated trafficking to sites of distant metastasis prior to the formation of detectable metastases in those sites. Through bulk and single-cell TCR sequencing of the Treg repertoire we reveal how LN colonization alters the clonal architecture of the Treg compartment and demonstrate how the evolution of metastatic traits coincides with shifts in the Treg repertoire. Parallel analyses in patients with melanoma and head and neck cancer reveal similar CD4 T cell dynamics in the context of LN involvement. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immune tolerance and metastatic progression. Citation Format: Cort B. Breuer, Norma A. Gutierrez, Zhewen Xiong, Amanda R. Kirane, John B. Sunwoo, Nathan E. Reticker-Flynn. Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR002.

Abstract C013: Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes

Abstract Introduction A sentinel lymph node is the primary node draining the tumor and is likely affected early in the metastatic process. The sentinel node holds a key position in the immune response against tumor in breast and represents a unique connection between the tumor and the host immune response. Detection of lymph node metastasis is important for staging a patient’s disease and is closely associated with prognosis. Although the clinical significance is debated, isolated tumor cells (<0.2 mm) appear to have no impact on survival while macro-metastasis (>2 mm) are linked to a poorer prognosis. Alterations in the immune response in lymph nodes with large versus small metastases have been observed, but little is known about the relationship between the immune response in the primary tumor and the metastatic status of the corresponding lymph nodes. Methods We analyzed single-cell suspensions from primary tumors and lymph nodes of 38 patients with early, operable breast cancer enrolled in the OSLO2 clinical observational trial using mass cytometry with a 47- antibody immune panel (including PanKeratin and EpCAM). In total 3.7 million single cells were analyzed and clustered into 40 metaclusters using FlowSOM. A second set of 22 metastatic lymph nodes from the OSLO2 cohort was used to validate results form the metastatic tumor cells. Additionally, immunofluorescence with E-cadherin and PanKeratin was applied, and intensity measured in 4 metastatic lymph nodes and imaging mass-cytometry was applied on one metastatic lymph node Results We found that the immune microenvironment in primary tumor was dominated by CD4 and CD8 T cells, particularly of exhausted and memory phenotype and with a higher frequency of activated regulatory T cells (Treg). In contrast, the immune composition in lymph nodes was predominantly composed of naturally occurring naive B and T cells. Notably, in line with previous publications we observed a skewing towards memory and exhausted phenotype in T cells, along with increased abundance of activated Treg in lymph nodes with larger metastasis to those with smaller or no metastasis. Furthermore, there were no correlation between the immune profile in the primary tumor and corresponding lymph node. The immune profile in the primary tumor was influenced by tumor size, with larger tumors showing a higher frequency of activated Tregs and exhausted CD8 T cells. Furthermore, In situ analysis revealed that the microenvironment in smaller metastase had an mesenchymal phenotype, while larger tumor deposits exhibited a more epithelial-like phenotype. ConclusionThe immune profile of the primary tumor did not predict the immune profile or metastatic status of the corresponding lymph node. Tumor size in both primary tumors and metastatic lymph nodes is the main drivers of changes in immune cell composition. Tumor cells from smaller metastases exhibited an mesenchymal phenotype, while larger metastasis displayed an epithelial phenotype. Citation Format: Inga Hansine H. Rye, Marit Otterlei Fjørtoft, Inger Riise Bergheim, Karin Teien Lande, Kanutte H. Huse, June Helen H. Myklebust, Ole Christian H. Lingjærde, Øystein. Garred, Jon H. Lømo, Hege H. Russnes. Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C013.

Abstract A004: E2F5 conditional knockout in mammary epithelium drives organotropic metastasis in breast cancer

Abstract Breast cancer ranks as the most commonly diagnosed cancer in women with 90% of associated deaths caused by distant metastasis. The metastatic lesions in brain, lung, liver, bone and lymph nodes in human breast cancer are not arbitrary, but rather are a complex mechanism of extrinsic and intrinsic regulatory factors controlling organ-specific colonization. Specialized mechanisms driving bone, brain and lung cancer have been described. However, the studies of liver and lymph node metastatic drivers are limited despite the prevalence of metastases in these organs. A significant limitation is the lack of genetically engineered mouse models that develop mammary tumors that spontaneously metastasize to liver and lymph nodes. Our previous research identified the role for E2F5 in both mammary gland development and breast cancer. Loss of E2F5 resulted in tumorigenesis after a latency of one year and 70% of mice showed metastatic lesions in lung, lymph node and liver. Transplantation of these spontaneous tumors into FVB MMTV Cre mice recapitulated the metastatic pattern observed in the primary tumors. We propose that E2F5 regulates drivers of metastasis and potentiates organ tropism to lymph nodes and the liver. To address this hypothesis, we conducted serial transplantations of tumors originating from the lymph nodes and liver back into the mammary gland. This enrichment study led to a significant increase in organ-specific metastasis, with 60% and 77% of penetrance observed in two distinct mouse lineages with liver (LVM) and lymph node tropism (LNM). Immunohistochemical studies confirmed a complete epithelial-mesenchymal transition (EMT) phenotype in LVM that is conserved in the cell lines generated. LNM showed a partial EMT with a more epithelial phenotype in cell lines. We observed the presence of an immune enriched environment in primary and metastatic tumors in both lineages. Bulk-RNA sequencing studies uncovered the presence of unique organotropic pathways associated with liver and lymph node metastasis. We observed enriched pathways from the primary tumor as major component of our organotropic drivers with a clear distinction between liver and lymph node enriched lineages. Additionally, a gradual acquisition of distant organ-specific markers and signaling pathways suggests that phenotypic mimicry and tumor plasticity are key for successful events of colonization to liver and lymph nodes in our model. With these results, we unravel potential mechanisms responsible for the organotropic metastases in breast cancer under the absence of the transcription factor E2F5. Citation Format: Jesus A. Garcia Lerena, Briana To, Jing-Ru Jhan, Eran Andrechek. E2F5 conditional knockout in mammary epithelium drives organotropic metastasis in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A004.