Node Sampling Research Articles

CSChighE-cadherinlow immunohistochemistry panel predicts poor prognosis in oral squamous cell carcinoma

Identifying marker combinations for robust prognostic validation in primary tumour compartments remains challenging. We aimed to assess the prognostic significance of CSC markers (ALDH1, CD44, p75NTR, BMI-1) and E-cadherin biomarkers in OSCC. We analysed 94 primary OSCC and 67 metastatic lymph node samples, including central and invasive tumour fronts (ITF), along with clinicopathological data. We observed an increase in ALDH1+/CD44+/BMI-1- tumour cells in metastatic lesions compared to primary tumours. Multivariate analysis highlighted that elevated p75NTR levels (at ITF) and reduced E-cadherin expression (at the tumour centre) independently predicted metastasis, whilst ALDH1high exhibited independent predictive lower survival at the ITF, surpassing the efficacy of traditional tumour staging. Then, specifically at the ITF, profiles characterized by CSChighE-cadherinlow (ALDH1highp75NTRhighE-cadherinlow) and CSCintermediateE-cadherinlow (ALDH1 or p75NTRhighE-cadherinlow) were significantly associated with worsened overall survival and increased likelihood of metastasis in OSCC patients. In summary, our study revealed diverse tumour cell profiles in OSCC tissues, with varying CSC and E-cadherin marker patterns across primary tumours and metastatic sites. Given the pivotal role of reduced survival rates as an indicator of unfavourable prognosis, the immunohistochemistry profile identified as CSChighE-cadherinlow at the ITF of primary tumours, emerges as a preferred prognostic marker closely linked to adverse outcomes in OSCC.

Quicker Path planning of a collaborative dual-arm robot using Modified BP-RRT* algorithm

Path-planning of an industrial robot is an important task to reduce the overall operation time. In industrial tasks, path planning is executed with lead-through programming, where in most cases the robot faces singulated object configurations. Cluttered environments demand path-planning algorithms, which are sensor driven, rather than pre-programmed. Path-planning algorithms, like RRT, and RRT* and their variants have inherent problems like the duration of a search and the creation of several node samples which may lead to longer path lengths. Back Propagation-Rapidly exploring Random Tree* (BP-RRT*) algorithm was a leap forward when an obstacle is enveloped with a sphere. Due to the spherical envelope of the obstacle, this method evaluates the connection between the path and obstacle in space with a spherical envelope using the triangular function and identifies the non-collision path in 3D space. This predicts the best non-collision path in the 3D workspace. The current state-of-the-art of BP-RRT* is limited to single-arm robots. A collaborative dual-arm robot faces more problems in path planning than a single-arm robot like inter-collision of manipulator arms apart from avoiding obstacles. A Modified BP-RRT* algorithm is proposed for the dual-arm collaborative robot has a pre-stage partition of grids that makes the computation faster, efficient, and collision-free compared to the traditional path planning algorithms namely RRT, RRT*, Improved RRT* and BP-RRT*. The algorithm is implemented in simulation as well as in physical implementation for ABB YuMi dual-arm collaborative robot and the typical length of the path of the proposed modified BP-RRT* method has reduced by 53.8% from the traditional RRT method, 6.95% from the RRT* method, 7.77% from improved RRT* method and 6.83% from the BP-RRT* method. Also, the average time to grasp has reduced by 17.84%, the typical duration for search has decreased by 33.45%, the number of node samples created has reduced by 14.79% from BP-RRT* algorithm.

Abstract PS05-03: MagSense® HER2, a Molecularly Targeted Magnetic Resonance Imaging Agent for the Detection of Axillary Nodal Metastasis in Subjects with Human Epidermal Growth Factor Receptor 2 Positive (HER2+) Breast Cancer

Abstract Background Precise nodal staging is critical in guiding systemic and regional treatments including surgery in the management of breast cancer. Regional nodal assessment includes axillary imaging (mostly by ultrasound) and lymph node sampling. Since imaging findings suggestive of nodal metastasis are based on size and morphologic changes and not specific to tumor type, pathologic confirmation through either biopsy or removal of sentinel lymph node (SLN) remains the gold standard. Imagion is developing the MagSense® HER2 Imaging Agent (MSH2IA), an anti-human epidermal growth factor receptor 2 (HER2) conjugated with iron oxide nanoparticles, for the detection of nodal metastasis in HER2+ breast cancer patients. MSH2IA is currently being investigated in a first-in-human phase 1 study (ACTRN12621000126819) in HER2+ breast cancer patients who are diagnosed to have suspicious nodes by conventional imaging. Methods The key objectives of the phase 1 study are safety and tolerability of MSH2IA and confirmation that MSH2IA drains to the axillary lymph nodes. The study also explores MSH2IA’s ability to detect metastatic nodes when used with magnetic resonance imaging (MRI), with tumor status assessed by standard tissue histopathology. All eligible subjects receive a 22.5 mg (iron equivalent) subareolar or peritumoral injection of MSH2IA. MRI of the axilla are obtained before and 24 to 72 hours after MSH2IA injection. Core biopsy or dissection of a node suspicious by ultrasound is obtained for histopathology assessments. After completing the first cohort of 6 subjects, the protocol was amended to include the insertion of an MRI compatible clip in a node suspicious by ultrasound to localize the suspicious node for core biopsy and postdose MRI scan. Review of MRI scans and histopathology are performed in respective central laboratories. Results Thirteen participants with HER2+ breast cancer have completed the study. MSH2IA appears safe and well tolerated. The imaging agent, as administered, is detectable by MRI and pathology, confirming that MSH2IA drains to the lymph nodes via the chosen route of administration. In postdose MRI scans, study central radiologists reported distinct and differentiable MRI appearance in morphologically normal and suspicious nodes. The presence of a distinct postdose MR image appearance was confirmed by a group of independent radiologists in a separate blinded review. While normal nodes appeared with homogeneous hypointensity, morphologically suspicious nodes showed partial irregular darkening and/or speckled heterogeneous hypointensity. In evaluable subjects, histopathology of the core biopsies obtained from the clipped nodes confirmed the presence of HER2+ nodal metastasis, providing preliminary evidence for node-level concordance between MRI vs pathology observations. Safety, imaging, and pathology results from all available subjects will be presented. This phase 1 study in Australia will be closing enrollment by July 31, 2023. A phase 2 study in the United States and elsewhere is being planned with the objectives of optimizing dose, schedule, and imaging parameters for best diagnostic performance for clinical utility. Conclusion Available data from the ongoing phase 1 study show that MSH2IA appears safe and drains to the axillary lymph nodes. MSH2IA displays a distinct and differentiable MRI appearance in nodes morphologically highly suspicious for tumor, which is confirmed to be HER2+ metastasis by histopathology by node level concordance. This data provides preliminary proof of principle for the mechanism of molecularly targeted MR imaging and suggests that MSH2IA has the potential to provide tumor-specific MR imaging not currently available in conventional axillary imaging. Citation Format: Jane Fox, Shanta Velaiutham, Natalie Yang, Eddie lau, Kowsi Murugappan, Beena Kumar, Alex Laslowski, Koushik Govindarajan, Steven Reich, Stephen Thomas, Nitika Neha, Marie Zhang, Isaac Bright. MagSense® HER2, a Molecularly Targeted Magnetic Resonance Imaging Agent for the Detection of Axillary Nodal Metastasis in Subjects with Human Epidermal Growth Factor Receptor 2 Positive (HER2+) Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS05-03.

Abstract PO3-23-11: Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). A significant number develop endocrine therapy resistance (ETR) . Clones develop in lymph nodes (LNs) which differ from the primary and these are implicated in ETR. A multiomics analysis of primary ER+ breast cancers, matched LN metastasis (LN+), if present, and matched recurrences (primary, nodal or distant) has been performed. Patients and Methods: The patients included are: Cohort A: 95 patients with matched primary and recurrent ER+ BC on different endocrine therapies. 35% had involved LNs at diagnosis. Recurrences: local in 59/95, concurrent local and nodal in 33/95, LN-only in 3/95. Median time to recurrence 4.5 years. Cohort B: 18 patients with ER+ LN+ BC who developed multiple recurrences (local and/or nodal, >3 consecutive recurrences/patient) on different lines of sequential endocrine therapy. Cohort C: 7 patients with ER+ breast cancer with multiple involved LNs treated with 3-6 months of neoadjuvant ET before surgery. Biopsies of primary and matched LNs performed at diagnosis and throughout treatment (3-4 timepoints/patient). Clinical response by serial ultrasound. Primary and LN samples (mean 12 samples/patient) were analysed together with normal DNA. Targeted RNAseq and DNA exome sequencing and whole-genome expression analysis was performed. Somatic mutations and copy number alterations (CNA) were determined, and differential gene expression analysis was performed. Validation of pathways implicated in ETR was by spatial NanoString GeoMx. All patients have long term follow-up. Results: Cohort A: A mean of 6 potential driver mutations identified by DNA sequencing in each primary BC (range 1-16). Most commonly mutated genes were PIK3CA, TP53, ERBB4, MAP3K1 and ERBB3. Multiple aberrations and a highly diverse mutational landscape were observed in all recurrences. In the majority of recurrences significant changes in variant allele frequency (VAF) were identified across several ETR-implicated genes and in over 50% new gains/losses were identified. Most frequently, losses were seen in ERBB3, ARID1A, HRAS and FOXA1 with gains in CDH1, NF1, PIK3CA, ESR1, TBX3 and GATA3 in recurrences vs primaries. Transcriptomic analysis revealed differentially expressed pathways including ER, HER2, GATA3, AKT, RAS and p63 signalling. Integrated analysis revealed several driver mutations with corresponding changes in upstream pathway activity. 33 patients had concurrent local and nodal recurrences and 52% had similar somatic mutation profiles in both with only minor variance in VAF. In 48%, 1 or more new potential driver mutations was identified in axillary nodes compared to recurrent primary cancers with losses in AKT1, CDH1 and TP53 being most common. Multiomics profiling of Cohorts B and C is complete and analysis underway. Discussion: This multiomics study is the largest cohort to-date of matched early and recurrent ER+/HER2- cancers. It sheds new light on the complex somatic and transcriptomic changes in local and nodal recurrent disease. The mechanisms of ETR are diverse and underlying mechanisms and clinically meaningful biomarkers including potentially actionable mutations and targets have been identified. Cohort C will uniquely capture and compare clonal evolution of breast cancer in primary and multiple matched lymph during neoadjuvant ET. Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Lorna Renshaw, J Michael Dixon, Arran K Turnbull. Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-11.

Abstract PO1-07-08: Value of high resolution full field optical coherence tomography and dynamic cell imaging (D-FFOCT) for one-stop rapid diagnosis breast clinic

Abstract BACKGROUND: Full field optical coherence tomography combined to dynamic cell imaging (D-FFOCT) is a new, simple to use, nondestructive, quick technique than can provide sufficient spatial resolution to mimic histopathological analysis. The objective of this study was to evaluate diagnostic performance of D-FFOCT for one-stop rapid diagnosis breast clinic. METHODS: D-FFOCT was applied to fresh untreated breast and nodes biopsies. Four different readers (senior and junior radiologist, surgeon and pathologist) analyzed the samples without knowing final histological diagnosis or ACR classification. The results were compared to conventional processing and staining (Hematoxylin-eosin). RESULTS: A total of 217 biopsies were performed on 152 patients. There were 144 breast biopsies and 61 lymph nodes with 101 infiltrative cancers (49,27%), 99 benign lesions (48,29%), 3 ductal in situ carcinoma (1,46%) and 2 atypias (0,98%). The diagnostic performances results were as follow: sensitivity: 77% [0.7;0.82], specificity: 64% [0.58;0.71], PPV: 74% [0.68;0.78] and NPV: 75% [0.72;0.78]. A large images atlas was created as well as a diagnosis algorithm from the readers experience. CONCLUSION: D-FFOCT provides interesting results defining malignancy on fresh breast and nodes samples. By training with the diagnosis algorithm and the images atlas or by using machine learning, radiologists could obtain even better outcomes allowing quick detection of breast cancer and lymph node involvement. diagnosis algorithm Figure 1 Images in DCI and FFOCT Two infiltrating cancer diagnosed on pathology (A,B). Focusing on cells (C). Normal galactophoric duct (D). Adenofibroma (E) Citation Format: Alexis Simon, Yasmina Badachi, Jacques Ropers, Isaura Laurent, Lida Dong, Elisabeth Da Maia, Agnes Bourcier, Geoffroy Canlorbe, Catherine Uzan. Value of high resolution full field optical coherence tomography and dynamic cell imaging (D-FFOCT) for one-stop rapid diagnosis breast clinic [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-08.

Abstract PO1-13-03: Tumour infiltrating double negative (CD20+CD27- IgD-) B cells in high-risk early breast cancers. Friend or Foe??

Abstract Background: B cells exhibit diverse phenotypes and function and the complex interplay between different B cell subsets in the context of chemotherapy treated breast cancers remains unclear. Here, we investigate the dynamic changes in the B cell immune landscape before and after NACT treatment across different breast cancer subtypes. Methods: Treatment naïve, mid-treatment and post-NACT breast tumour, matched lymph node, blood and serum samples were profiled for B cell subsets and cytokines by flow cytometry, cytometry by time-of-flight (CyTOF) and Luminex technologies. Ex vivo autologous tumour organoid and immune cells cocultures to functionally assess the B-cell interactions across breast cancer subtypes were performed. Multiplex spatial analysis method was used to explore the dynamics of double negative B cell cellular crosstalk within the tumour and axillary lymph nodes. The results are being associated with treatment outcomes. Expression and activity of purinergic ectoenzymes within B cell subsets were also assessed by flow cytometry and high-performance liquid chromatography. Current work focusses on how these findings may relate to immunotherapy response, a new paradigm shift in the treatment of triple negative breast cancer patients. Results: We observed a significant expansion in the CD20+CD27−IgD− cells, also termed as double-negative (DN) B cells, within the TME of treatment naïve and post-neoadjuvant residual tumours compared to the periphery. Specifically, DN1 (CD20+ CD27−IgD− CD21+CXCR5+) cells represented the majority of DN B cells within the treatment naïve TME and in circulation. However, NACT promotes an expansion of the DN2 (CD20+CD27−IgD−CD21-CXCR5-) subset as well as a relatively uncharacterised DN3 (CD19+IgD−CD27−CXCR5−CD11c−) cell population. Preliminary analyses on how these changes (especially the enrichment of DN2 cells) relate to treatment response show significant expansion of DN2 cells in the periphery of poor responders to NACT, coupled with significantly reduced infiltration of DN2 into resistant residual tumours. Among circulating B, T and NK cells, B cells showed the highest CD73 and CD39 surface expression at baseline, but their expression significantly drops following NACT especially within the DN2 B cell subset. These ecto-5’-nucleotidases are known to play a critical role in B and T cell interactions. Spatial analyses between these cell types is ongoing. Conclusion: We report for the first time an enrichment of DN cells in breast cancer tumour tissue, specifically the expansion of DN2 cells following neoadjuvant chemotherapy. Functional analyses of tumour-infiltrated B-cells suggest that mechanistically, these B-cell subgroups may contribute to immunosurveillance and point to an important role of purinergic signalling in early breast cancers. Our study highlights the requirement for further investigation into the role of DN B cells in the context of chemo/immunotherapy resistance in breast cancers. Citation Format: Esme Carpenter, Paul Buckley, Thanussuyah Alaguthurai, Rosalind Graham, Helen Kakkassery, Gheed Alhity, Farhana Hossain, Sadiyah Mukhtar, Sheeba Irshad. Tumour infiltrating double negative (CD20+CD27- IgD-) B cells in high-risk early breast cancers. Friend or Foe?? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-03.

Abstract PO4-25-01: Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers

Abstract Lymph node metastasis is a prognostically significant factor in breast cancer, though the colonization mechanism of cancer cells in this immune cell-rich organ remains elusive. This study aimed to elucidate the impact of breast cancer on the lymph node immune cell landscape. Multiscale transcriptomic analyses were performed on both metastatic and non-metastatic lymph node samples from breast cancer patients with lymph node metastasis. Twenty laser-micro-dissected sections were obtained from 17 lymph nodes across six breast cancer patients at stages II–III, with each patient contributing both types of lymph nodes for direct comparison. Comparing the transcriptomes of paired lymph nodes with and without metastasis from the same patients revealed selective downregulation of CD169+ macrophage-related genes in metastatic lymph nodes. The spatial transcriptome indicated a potential depletion of CD169+ macrophages, initiators of anticancer immunity, from their residence (sinuses) in metastatic lymph nodes, while other principal immune cell types were unaltered. Mass spectrometry imaging revealed that the numbers of CD169+ macrophages were smaller in the metastatic lymph nodes than in the non-metastatic lymph nodes, suggesting that cancer cells uprooted CD169+ macrophages from the lymph nodes. Conversely, the count of B, T, Treg, and CD11c+ cells remained comparable in both lymph node types, albeit an enrichment of Treg cells around metastasized cancer tissues was observed. Additional immunohistochemistry analysis of 315 non-metastatic lymph nodes and 159 metastatic lymph nodes from 58 patients with breast cancer showed that a reduced CD169+ macrophage population was prevalent in various breast cancer subtypes. A subset of metastatic lymph nodes (37 out of 159) displayed a complete absence of detectable CD169+ macrophages. The data also depicted a gradual decline in CD169+ macrophages correlating with the pN classification, while no correlation was identified with pathological tumor size classification (pT) or metastasized cancer volume. The data suggest precedence of CD169+ macrophage elimination over other reported immune cell abnormalities, such as cell number and metabolic irregularities. CD169+ macrophages are a unique type of resident macrophages in the lymphoid organs that present cancer-derived antigens to CD8+ cells. The antigen-presenting role of CD169+ macrophages to T cells, a pivotal step in adaptive immunity, signifies the catastrophic implications of their suppression. This study underscores CD169+ macrophage suppression as a pronounced pathological phenotype in lymph nodes with breast cancer metastasis, thereby establishing it as a critical future therapeutic target. Citation Format: Yurina Maeshima, Tatsuki R. Kataoka, Alexis Vandenbon, Masahiro Hirata, Yasuhide Takeuchi, Yutaka Suzuki, Yukiko Fukui, Yumiko Ibi, Hironori Haga, Satoshi Morita, Masakazu Toi, Shinpei Kawaoka, Kosuke Kawaguchi. Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-01.

Sublobectomy and lymph node sampling are adequate for patients with invasive lung adenocarcinoma presenting as pure ground glass nodules.

In this study, we aimed to investigate the prognosis of invasive lung adenocarcinoma that manifests as pure ground glass nodules (pGGNs) and confirm the effectiveness of sublobectomy and lymph node sampling in patients with pGGN-featured invasive adenocarcinoma (IAC). We retrospectively enrolled 139 patients with pGGN-featured IAC, who underwent complete resection in two medical institutions between January 2011 and May 2022. Stratification analysis was conducted to ensure balanced baseline characteristics among the patients. The 5-year overall survival (OS) and disease-free survival (DFS) rates were compared between the groups using Kaplan-Meier survival curves and log-rank test. The 5-year OS and DFS rates for patients with IAC presenting as pGGNs after surgery were 96.5% and 100%, respectively. No lymph node metastasis or recurrence was observed in any of the enrolled patients. There was no statistically significant difference in the 5-year OS between patients who underwent lobectomy or sublobectomy, along with lymph node resection or sampling. IAC presented as pGGNs exhibited low-grade malignancy and had a relatively good prognosis. Therefore, these patients may be treated with sublobectomy and lymph node sampling.

Development and preliminary application of a quadruplex real-time PCR assay for differential detection of porcine circovirus 1-4 in Chengdu, China.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1-4 ORF2 gene to establish a PCV1-4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1-4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1-4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

Evaluation of Sensitivity and Specificity of Ultrasound-Guided FNA of Suspicious Axillary Lymph Nodes in Patients with Breast Cancer

Background: The axillary lymph node status in breast cancer is a major prognostic factor in survival and establishing a personalized treatment scheme. The ultrasound-guided fine needle aspiration (US-FNA) is a method for taking a lymph node sample. It allows physicians to decide how to manage the axilla. Objectives: This study was conducted to investigate the sensitivity of the US-FNA technique on suspicious axillary lymph nodes with a thickness of 3 to 6 mm in breast cancer patients. Methods: In a cross-sectional study, all the patients were subjected to preoperative ultrasound evaluation of the axilla to determine the presence of lymph nodes suspicious of malignancy. In cases where the suspicious lymph node cortex size was between 3 and 6 mm, US-FNA was performed. After surgery, the frozen section of the biopsy sample was examined histologically and compared with fine needle aspiration (FNA) cytology results. Results: A total of 102 patients were examined in the study. FNA test results indicated that 46 subjects had axillary malignant tissue, and benign cases summed 56. Also, the final results of frozen section surgical histopathology identified 46.1% of patients with involved lymph nodes. The sensitivity and specificity of FNA were 93.62% and 96.36%, respectively. Also, the overall diagnostic accuracy was 95.1%. Conclusions: This study showed that the sensitivity, specificity, and accuracy were more than 90% for the ultrasound-guided FNA test in identifying involved lymph nodes in patients with breast cancer. Therefore, the results of this test can be considered clinically reliable. However, there is still a need to examine the sensitivity and specificity of this method in identifying lymph node involvement.

GAN-based self-supervised message passing graph representation learning

Graph representation learning aims to learn embedding representations of nodes in graph-structured data through supervised or unsupervised methods. However, for unlabeled or sparsely labeled graph datasets, conventional supervised approaches fail to perform adequately, and existing unsupervised methods have difficulty achieving high accuracy on multiple downstream tasks such as node classification and link prediction at lower embedding dimensions. To address this issue, we propose a novel self-supervised graph representation learning approach called GAN-based Message Passing Graph Representation Learning. This approach significantly improves the accuracy of node embeddings without relying on label information. Specifically, GMP-GL has developed a generator based on neighbor-weighted random walk to obtain positive and negative node sample pairs, as well as a discriminator that distinguishes whether a real connection exists between node pairs. Through adversarial learning, GMP-GL automatically learns node embeddings. Furthermore, GMP-GL optimizes the training outcomes of the generative adversarial model through self-supervised embedding aggregation learning, thus introducing rich structural information from the graph for the subsequent rounds of generative adversarial training. In addition, GMP-GL employs multiple autoencoders to generate and filter original embeddings, ensuring that the pre-embeddings preserve a significant amount of original feature information for formal learning. The study selects six real-world datasets and conducts comparative experiments with ten SOTA baseline methods. The experimental results demonstrate that the proposed model not only surpasses previous unsupervised methods but also matches or even exceeds the performance of supervised methods. This indicates the great potential of the proposed approach for representation learning tasks on unlabeled graph datasets and its effective applicability to real-world problems.

A novel use of endobronchial ultrasound bronchoscope to biopsy supraclavicular lymph node

Background: Accurate staging for patients with lung cancer is essential for management and prognostication. Percutaneous ultrasound-guided fine needle aspiration (US FNA) of supraclavicular lymph nodes is not always performed clinically for variety of reasons. Our study explores the utility of supraclavicular lymph node biopsy using the endobronchial ultrasound (EBUS) bronchoscope at the time of a mediastinal staging procedure as a possible alternative to US FNA. Methods: This study included 22 patients who underwent supraclavicular lymph node biopsy using the EBUS bronchoscope during lung cancer staging procedure from 2019 to 2021 at the University of Pennsylvania. Feasibility and safety of the procedure were reported. Descriptive statistics were performed for baseline demographics, imaging features, pathology results, tumor cellularity and adequacy for oncological testing. Results: The procedures were safely performed in all patients with no safety events reported. All patients were diagnosed with malignancy and EBUS-guided supraclavicular lymph node FNA showed definitive diagnosis in 19. Of the 16 patients with the final diagnosis of non-small cell lung cancer (NSCLC), 14 had tumor seen in supraclavicular lymph node sampling and 9 were upgraded to Stage IIIB disease from lower mediastinal lymph node staging. Eleven supraclavicular lymph node samples had tumor cellularity >10%. Eight samples were sent for next generation sequencing (NGS). Three of them were the only source for NGS. Conclusions: Supraclavicular lymph node biopsy using the EBUS bronchoscope is a feasible and safe procedure to perform during lung cancer staging. Sampling has significant clinical value for accurate staging and oncologic testing.

EnBloc Resection of Sentinel Lymph Nodes with the Hysterectomy Specimen in Endometrial Cancer.

We demonstrate the surgical technique of removing the sentinel lymph nodes with its afferent lymphatic vessels attached to the hysterectomy specimen. Stepwise demonstration of the technique with narrated video footage. Sentinel lymph node sampling has been established as an acceptable staging method in endometrial cancer cases.1 Lymphatic anatomy has been described according to three consistent channels for endometrial cancer dissemination: (1) an upper paracervical pathway draining external or obturator lymph nodes; (2) a lower pathway draining internal iliac lymph nodes; and (3) the infundibulo-pelvic pathway with a course along the broad ligament.2 A study in patients with cervical cancer identified tumor cells in the afferent lymphatic vessels of the upper pathway, even when the corresponding sentinel node was negative (3/20 patients).3 This could be an important prognostic factor in patients with cervical cancer. Since the typical position of sentinel nodes is the same in both endometrial and cervical cancers, we aimed to assess the feasibility of removing 'enbloc' the sentinel node with its afferent lymphatic vessels, and the uterus.4 INTERVENTIONS: The Da Vinci Xi surgical system was used. Indocyanine green was injected cervically, the pelvic surgical spaces were developed, and the sentinel lymph nodes, along with the afferent lymphatic vessels, were identified using the Firefly infrared camera. The lymphovascular tissue was mobilized and separated from the uterine artery, which was skeletonized and ligated. Colpotomy was performed and the specimen was retrieved vaginally. Emerging evidence regarding diagnosis, characterization, and treatment of endometrial cancer has introduced a new era, based on minimally invasive techniques for staging through sentinel lymph node biopsy, molecular classification, and personalized treatment algorithms that include immune checkpoint inhibitors and targeted therapies.5 Lymph node staging is one of the most significant prognostic factors in endometrial cancer patients and is a guide for adjuvant treatment. Sentinel lymph node biopsy is not inferior to conventional lymphadenectomy and is in fact a better way of identifying low-volume cancer through the use of ultrastaging, as part of the sentinel node algorithm.6 The dissection technique described in this video could offer an improvement in the staging of endometrial cancer, ensuring that the true sentinel lymph node is identified and that potential cancer cells inside the afferent lymphatic vessels are also excised. Therefore, it could be utilized as a more accurate way of planning adjuvant treatment and consequently improving recurrence and survival; however more studies are needed to further evaluate the feasibility and sensitivity of identifying disease in the afferent lymphatic vessels. This novel surgical technique emphasizes the importance of anatomical knowledge and offers inspiration for studies with potential clinical benefit that should follow.

A selective anatomically based lymph node sampling can replace a side specific pelvic lymphadenectomy in endometrial cancer with failed sentinel node mapping

AimTo evaluate the locations of metastatic pelvic sentinel nodes (SLN) and the proportion of SLNs outside and within defined typical anatomical positions along the upper paracervical lymphatic pathway (UPP). Patients and methodsConsecutive women with endometrial cancer (EC) of all risk groups underwent pelvic SLN-detection using cervically injected indocyanine green (ICG). A strict anatomically based algorithm and definitions of SLNs was adhered to. The positions of ICG-defined SLNs were intraoperatively depicted on an anatomical chart. All SLNs were examined using ultrastaging and immunohistochemistry. The proximal third of the obturator fossa and the interiliac area were defined as typical positions. The parauterine lymphovascular tissue (PULT) was separately removed. The proportions of metastatic SLNs, overall and isolated, typically, and atypically positioned were analyzed per woman. ResultsA median of two (range 1–12) SLN metastases along the UPP including the PULT were found in 162 women. 41 of 162 women (25.3 %) had isolated metastases in the obturator fossa harboring 49.1 % of all SLN metastases. Three women (1,9 %) had isolated PULT metastases. SLN metastases outside typical positions were identified in 28/162 women (17.3 %); isolated metastases were seen in seven women (4.3 %), so 95.7 % of pelvic node positive women had at least one metastatic SLN located at a typical position. ConclusionA selective removal of lymph nodes at typical proximal obturator and interiliac positions and the PULT can replace a full side specific pelvic LND when SLN mapping is unsuccessful. The obturator fossa is the predominant location for metastatic disease.

Lymph node dissection in lung cancer surgery.

Lung cancer, a leading cause of cancer-related death, often requires surgical resection for early-stage cases, with recent data supporting less invasive resections for tumors smaller than 2 cm. Central to resection is lymph node assessment, an area of controversy worldwide, compounded by advances in minimally invasive techniques. The review aims to assess current standards for lymph node assessment, recent data from the surgical era, and the immunobiological basis of how lymph node metastases impact patient outcomes. The British Thoracic Society guidelines recommend systematic nodal dissection during lung cancer resection, without specifying node removal or sampling. Historical data on mediastinal lymph node dissection (MLND) survival benefits are inconclusive, although proponents argue for lower recurrence rates. Recent trials such as ACOSOG Z0030 found no survival difference between MLND and nodal sampling, reinforcing the need for robust staging. While lobe-specific dissection strategies have been proposed, they currently lack consensus. JCOG1413 aims to compare the clinical benefits of lobe-specific and systematic dissection. TNM-9 staging revisions emphasize the prognostic significance of single-station N2 involvement. Robotic surgery shows promise, with trials such as RAVAL, which reported comparable outcomes to video-assisted thoracic surgery (VATS) and improved lymph node sampling. Immunobiological insights suggest preserving key immunological sites during lymphadenectomy, especially for patients receiving adjuvant immunotherapy. In conclusion, the standard lymph node resection strategy remains unsettled. The debate between systematic and selective dissection continues, with implications for staging accuracy and patient outcomes. As minimally invasive techniques evolve, robotic surgery emerges as an effective and low-risk approach to delivering optimal lymph node assessment.

Small Non-Coding RNAs and Their Role in Locoregional Metastasis and Outcomes in Early-Stage Breast Cancer Patients.

Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.

CD20-позитивные B-лимфоциты в реактивном микроокружении опухоли как независимый иммуногистохимический фактор прогноза при нодулярном склерозе классической лимфомы Ходжкина

Aim. To assess the prognostic value of CD20-positive B-lymphocytes in the reactive tumor microenvironment using histological specimens of lymph nodes from patients with nodular sclerosis classical Hodgkin lymphoma (cHL).
 Materials & Methods. The analysis focused on archival samples (paraffin blocks and cassettes) of lymph nodes from 71 patients with newly diagnosed cHL, nodular sclerosis type. Immunohistochemical methods were used for morphometric assessment of CD20-positive cells in the tumor microenvironment. In each lymph node biopsy sample, 20 fields of view were analyzed for the mean relative count of CD20-positive B-lymphocytes in the reactive tumor microenvironment. The В-cell count was determined by the double-blind method. Patients were aged 18–77 years (median 35 years); there were 37 women and 34 men. According to the primary documentation, 11 (15.5 %) patients received ABVD as first-line therapy and 60 (84.5 %) patients were treated with BEACOPP-14 (EACOPP-14) or BEACOPPesc regimens.
 Results. A low count of CD20-positive B-lymphocytes in the reactive tumor microenvironment is regarded as an independent factor of poor prognosis in patients with nodular sclerosis cHL. This is the category of patients who show worse event-free survival (EFS) rates. In the cohort with the subthreshold count of CD20-positive B-lymphocytes in the reactive microenvironment, the median EFS was 38 months, whereas in the cohort with the above-threshold count it was not reached.
 Conclusion. The results of this study demonstrate the need to assess the relative count of CD20-positive B-lymphocytes in the reactive tumor microenvironment in patients with nodular sclerosis cHL at the primary diagnosis stage. There is every reason to believe that for this category of patients the incorporation of this immunohistochemical parameter into the existing predictive models is fully justified.

Biological control of the native endophytic fungus Pochonia chlamydosporia from the root nodule of Dolichos lablab on Fusarium wilt of banana TR4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense, Tropical Race 4 (TR4) is a soil-borne disease, and it is devastating. At present, the biological control using antagonistic microorganisms to mitigate TR4 is one of the best strategies as a safe and green way. Yunnan has abundant and diverse microbial resources. Using the dual-culture method, the antagonistic endophytic fungi against TR4 were isolated and screened from the root nodule of Dolichos lablab. The effect of the highest antagonistic activity strain on the morphology of the TR4 mycelium was observed using the scanning electron microscope. According to morphological characteristics and sequence analysis, the strain was identified. The biocontrol effect and plant growth promotion were investigated by greenhouse pot experiment. Using the confocal laser scanning microscope and the real-time fluorescence quantitative PCR, the dynamics of TR4 infestation and the TR4 content in banana plant roots and corms would also be detected. In this study, 18 native endophytic fungi were isolated from a root nodule sample of Dolichos lablab in the mulch for banana fields in Yuxi, Yunnan Province, China. The YNF2217 strain showed a high antagonistic activity against TR4 in plate confrontation experiments, and the inhibition rate of YNF2217 is 77.63%. After TR4 culture with YNF2217 for 7 days in plate confrontation experiments, the morphology of the TR4 mycelium appeared deformed and swollen when observed under a scanning electron microscope. According to morphological characteristics and sequence analysis, the strain YNF2217 was identified as Pochonia chlamydosporia. In the greenhouse pot experiment, the biocontrol effect of YNF2217 fermentation solution on TR4 was 70.97% and 96.87% on banana plant leaves and corms, respectively. Furthermore, YNF2217 significantly promoted the growth of banana plants, such as plant height, leaf length, leaf width, leaf number, pseudostem girth, and both the aboveground and underground fresh weight. Observations of TR4 infestation dynamics in banana roots and corms, along with real-time fluorescence quantitative PCR, verified that YNF2217 inoculation could significantly reduce the TR4 content. Therefore, YNF2217 as P. chlamydosporia, which was found first time in China and reported here, is expected to be an important new fungal resource for the green control of Fusarium wilt of banana in the future.

Abstract 3778: Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact

Abstract Introduction: Recent studies on disseminated cancer cells (DCCs) in Non-Small-Cell Lung Cancer (NSCLC) patients demonstrated their clinical relevance even in early tumor stages. However, their molecular and phylogenetic link to the primary tumor is currently unclear. Certain genetic alterations, such as TP53 or KRAS mutations, have been proposed to be clonal events and drivers of dissemination and metastasis. In this study, we analyzed the genome and transcriptome of single DCCs to identify the characteristics of early DCCs and their translational clinical value. Materials and methods: From 2011 to 2019 we prospectively collected and analyzed bone marrow aspirates and lymph node samples from 296 newly diagnosed NSCLC patients undergoing tumor resection with curative intention. Presence of DCC was examined by i) immunocytology (IC) for Cytokeratin (clone A45-B/B3, bone marrow, BM-DCCs) or EpCAM (clone BerEp4, lymph node, LN-DCCs) and ii) immunofluorescent (IF) staining for EpCAM in a live cell suspension (clone HEA-125) from BM or LN. Single cells were isolated using micromanipulation and subjected to Whole Genome Amplification or combined Whole Genome/Transcriptome Amplification for IC and IF, respectively. Sanger sequencing for known hotspot mutations in TP53 and KRAS was performed on single DCCs. Gene expression profiles of DCCs were analyzed by single cell RNA sequencing and the results were compared with clinical data with a mean follow-up of 2.93 years (range 0.03-7.22). Results: LN-DCCs were found in 44.8% and 59.6% by IC and IF, respectively. CKIC pos BM-DCCs were found in 45.2%, while EpCAMIF pos BM-DCCs were present in 52.8%. LN-DCCs correlated with reduced tumor-specific survival (TSS, p=0.009) and progression-free survival (PFS, p=0.025). EpCAMIF pos, but not CKIC pos BM-DCCs correlated with reduced TSS (p=0.003) and PFS (p=0.083). Genetic analysis of single DCCs revealed that TP53 or KRAS mutations could be found in 3.6% and 2.8% of analyzed BM-DCCs and 5.0% and 29.3% of LN-DCCs for IF and IC, respectively. Single cell RNA-seq showed distinct transcriptomic profiles for LN- and BM-DCCs, and identified a DCC phenotype with high stemness score in the bone marrow. Further analysis of potential transcriptome-based biomarkers identified a novel candidate marker for activated EpCAMpos BM-DCCs that strongly correlates with PFS and TSS (p<0.001). Conclusions: DCCs in early-stage NSCLC have high prognostic value and predict reduced PFS and TSS. While genetic alterations in the TP53 and KRAS gene were not detected in the majority of analyzed DCCs, transcriptomic phenotypes provided relevant information and identified a subset of activated DCCs that correlate strongly with relapse and poor outcome. Citation Format: Tobias Mederer, Daniel Spitzl, Felix Elsner, Bernhard Polzer, Michael Ried, Reiner Neu, Tobias Robold, Hans-Stefan Hofmann, Christoph A. Klein. Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3778.

Abstract 5828: The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution

Abstract Progressive disease (PD) in patients with chronic lymphocytic leukemia (CLL) on ibrutinib often presents with acquired mutations in BTK and/or PLCG2. Reported variant allele frequencies (VAF) are often low, leading some to question the role of these mutations. Further, several co-existing mutations are often identified. Here we investigated the clonal composition of PD on single cell level, across time, and different anatomic compartments. 84 CLL patients (52 treatment-naïve; 32 relapsed/refractory) with either a TP53 aberration or age ≥65 years received single-agent ibrutinib 420 mg once daily until PD or limiting side effects on a phase 2 study (NCT01500733). The median progression-free survival was 86.6 months. 39 (46.4%) patients progressed, 31 with CLL (PD-CLL), and 8 with Richter’s transformation. Median time to PD-CLL was 57.2 months. In PD-CLL patients, peripheral blood (PB) was sent for genotyping of known hotspot mutations in BTK/PLCG2 (Neogenomics). We then designed digital droplet PCR probes to test for BTK C481S/R/Y and PLCG2 R665W/L845/S707F mutations in samples collected at baseline, during response to ibrutinib and at PD in 29 patients. 26 (89.7%) patients had detectable BTK/PLCG2 mutations (VAF range 0.04 - 0.96). 12 (41%) patients carried multiple BTK/PLCG2 mutations. In 11 evaluable cases single-cell DNA sequencing (Mission Bio) showed individual mutations present in separate cells consistent with branching evolution. Having confirmed oligoclonality, we calculated the cumulative cell fraction (cCF) of BTK/PLCG2 mutations in the 29 PD-CLL patients. 16 (55%) patients had cCF >0.5 (range 0.59-0.96), 10 patients (35%) had cCF < 0.5 (range 0.04-0.43) and 3 patients (10%) had no detectable mutations. BTK/PLCG2 mutations were detected in samples collected at median 21.6 months (range 0 - 75) prior to PD. In 11 cases, we tested lymph node (LN) biopsies at PD. In 6 cases with cCF <0.5 in PB, 4 corresponding LN samples had higher cCF (0.01/0.92; 0/0.85; 0.06/0.69; 0.46/0.99 for PB/LN, respectively). In 5 cases, PB contained 2-6 distinct subclones with cCF >0.5, in 3 cases the corresponding LN harbored fewer subclones than PB (6/3; 3/2; 3/0 for PB/LN, respectively). To characterize the genomic background of PD-CLL, we performed whole-exome sequencing of longitudinal samples from 26 patients. In addition to BTK/PLCG2, the most frequently mutated genes were TP53, SF3B1, NOTCH1, and POT1. Furthermore, copy number variations (CNA), such as del17p, del13q, del14q, del3p, and del8p, were common. In summary, in our cohort BTK/PLCG2 mutations were identified in 89.7% of PD-CLL patients. These mutations arise in oligoclonal populations with variable distribution across anatomical sites. In rare patients without BTK/PLCG2 mutations, the co-evolution of putative driver mutations and multiple CNAs generated genomic complexity reminiscent of lymphoma transformation. Citation Format: Chingiz Underbayev, Jonathan Chen, Hailey M. Harris, Lauren T. Vaughn, Andy Itsara, Adrian Wiestner. The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5828.