Open-ST: High-resolution spatial transcriptomics in 3D.

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

The Lymphatic Vasculature in the 21st Century: Novel Functional Roles in Homeostasis and Disease.

Mammals have two specialized vascular circulatory systems: the blood vasculature and the lymphatic vasculature. The lymphatic vasculature is a unidirectional conduit that returns filtered interstitial arterial fluid and tissue metabolites to the blood circulation. It also plays major roles in immune cell trafficking and lipid absorption. As we discuss in this review, the molecular characterization of lymphatic vascular development and our understanding of this vasculature's role in pathophysiological conditions has greatly improved in recent years, changing conventional views about the roles of the lymphatic vasculature in health and disease. Morphological or functional defects in the lymphatic vasculature have now been uncovered in several pathological conditions. We propose that subtle asymptomatic alterations in lymphatic vascular function could underlie the variability seen in the body's response to a wide range of human diseases.

Neuronal substance P drives metastasis through an extracellular RNA-TLR7 axis.

Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis.

Lymphatic vessels in cancer.

The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules, and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.

Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs.

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

The new (Version 9) American Joint Committee on Cancer tumor, node, metastasis staging for cervical cancer.

The American Joint Committee on Cancer (AJCC) tumor, node, metastasis (TNM) staging for all cancer sites has been periodically updated as a published manual for many years. The last update, the eighth edition AJCC Cancer Staging Manual went into use on January 1, 2018. The AJCC has since restructured and updated its processes, and all AJCC staging-related data are now housed on its new application programming interface. Consequently, the next AJCC TNM staging update, AJCC version 9 TNM staging, will be published electronically and will be released chapter by chapter. The first chapter of version 9 AJCC TNM staging is the updated cervical cancer staging, which is now published. This article highlights the changes to the AJCC TNM cervical cancer staging; these changes align with the International Federation of Gynecology and Obstetrics staging. The most important of the changes are: 1) the incorporation of imaging and surgical findings, 2) the elimination of lateral spread from T1a, 3) the addition of a subcategory to T1b (T1b3), and 4) histopathology is updated to reflect human papillomavirus-associated and human papillomavirus-independent carcinomas.

A CXCL1 paracrine network links cancer chemoresistance and metastasis.

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

Melanoma-derived mediators can foster the premetastatic niche: crossroad to lymphatic metastasis.

The natural history of advanced malignant melanoma demonstrates that, in most cases, widespread tumor dissemination is preceded by regional metastases involving tumor-draining lymph nodes [sentinel lymph nodes (SLNs)]. Under physiological conditions, LNs play a central role in immunosurveillance to non-self-antigens to which they are exposed via afferent lymph. The dysfunctional immunity in SLNs is mediated by tumor secretory factors that allow the survival of metastatic melanoma cells within the LN by creating a premetastatic niche (PMN). Recent studies outline the altered microenvironment of LNs shaped by melanoma mediators. Here, we discuss tumor secretory factors involved in subverting tumor immunity and remodeling LNs and highlight emerging therapeutic strategies to reinvigorate antitumoral immunity in SLNs.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

Nonmetastatic Axillary Lymph Nodes Have Distinct Morphology and Immunophenotype in Obese Patients with Breast Cancer at Risk for Metastasis.

Obese patients with breast cancer have worse outcomes than their normal weight counterparts, with a 50% to 80% increased rate of axillary nodal metastasis. Recent studies suggest a link between increased lymph node adipose tissue and breast cancer nodal metastasis. Further investigation into potential mechanisms underlying this link may reveal potential prognostic utility of fat-enlarged lymph nodes in patients with breast cancer. This study used a deep learning model to identify morphologic differences in nonmetastatic axillary nodes between obese, node-positive, and node-negative patients with breast cancer. The model was developed using nested cross-validation on 180 cases and achieved an area under the receiver operator characteristic curve of 0.67 in differentiating patients using hematoxylin and eosin-stained whole slide images. The morphologic analysis of the predictive regions showed an increased average adipocyte size (P = 0.004), increased white space between lymphocytes (P < 0.0001), and increased red blood cells (P < 0.001) in nonmetastatic lymph nodes of node-positive patients. Preliminary immunohistochemistry analysis on a subset of 30 patients showed a trend of decreased CD3 expression and increased leptin expression in fat-replaced axillary lymph nodes of obese, node-positive patients. These findings suggest a novel direction to further investigate the interaction between lymph node adiposity, lymphatic dysfunction, and breast cancer nodal metastases, highlighting a possible prognostic tool for obese patients with breast cancer.

Oleic acid-PPARγ-FABP4 loop fuels cholangiocarcinoma colonization in lymph node metastases microenvironment.

BACKGROUND AND AIMS: Lymph node metastasis is a significant risk factor for patients with cholangiocarcinoma, but the mechanisms underlying cholangiocarcinoma colonization in the lymph node microenvironment remain unclear. We aimed to determine whether metabolic reprogramming fueled the adaptation and remodeling of cholangiocarcinoma cells to the lymph node microenvironment. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing of primary tumor lesions and paired lymph node metastases from patients with cholangiocarcinoma and revealed significantly reduced intertumor heterogeneity and syntropic lipid metabolic reprogramming of cholangiocarcinoma after metastasis to lymph nodes, which was verified by pan-cancer single-cell RNA sequencing analysis, highlighting the essential role of lipid metabolism in tumor colonization in lymph nodes. Metabolomics and in vivo CRISPR/Cas9 screening identified PPARγ as a crucial regulator in fueling cholangiocarcinoma colonization in lymph nodes through the oleic acid-PPARγ-fatty acid-binding protein 4 positive feedback loop by upregulating fatty acid uptake and oxidation. Patient-derived organoids and animal models have demonstrated that blocking this loop impairs cholangiocarcinoma proliferation and colonization in the lymph node microenvironment and is superior to systemic inhibition of fatty acid oxidation. PPARγ-regulated fatty acid metabolic reprogramming in cholangiocarcinoma also contributes to the immune-suppressive niche in lymph node metastases by producing kynurenine and was found to be associated with tumor relapse, immune-suppressive lymph node microenvironment, and poor immune checkpoint blockade response. CONCLUSIONS: Our results reveal the role of the oleic acid-PPARγ-fatty acid-binding protein 4 loop in fueling cholangiocarcinoma colonization in lymph nodes and demonstrate that PPARγ-regulated lipid metabolic reprogramming is a promising therapeutic target for relieving cholangiocarcinoma lymph node metastasis burden and reducing further progression.

Single-cell RNA sequencing reveals the heterogeneity of epithelial cell and fibroblast cells from non- to metastatic lymph node OTSCC.

Lymph node metastasis (LNM) is one of the common features of oral tongue squamous cell carcinoma (OTSCC). LNM is also taken as a sign of advanced OTSCC and poor survival rate. Recently, single-cell RNA sequencing has been applied in investigating the heterogeneity of tumor microenvironment and discovering the potential biomarkers for helping the diagnosis and prognosticating. Pathogenesis of LNM in OTSCC remains unknown. Specifically, cancer-associated fibroblasts (CAFs) and epithelial tumor cells could foster the progression of tumors. Thus, in this study, we aimed to comprehensively analyze the roles of subpopulations of CAFs and epithelial tumor cells in lymph node metastatic OTSCC using the integration of OTSCC single-cell RNA sequencing datasets. Four distinct subtypes of CAFs, namely vascular CAFs, myofibroblast CAFs, inflammatory CAFs, and growth arrest CAFs were successfully discovered in LNM tumor and confirmed the roles of GAS and PTN pathways in the progression of tumor metastasis. In addition, NKAIN2+ epithelial cells and FN1+ epithelial cells specifically exhibited an upregulation of PTN, NRG, MIF, and SPP1 signaling pathways in the metastatic OTSCC. In doing so, we put forth some potential biomarkers that could be utilized for the purpose of diagnosing and prognosticating OTSCC during its metastatic phase and tried to confirm by immunofluorescence assays.

Differentiated and anaplastic thyroid carcinoma: Major changes in the American Joint Committee on Cancer eighth edition cancer staging manual.

Answer questions and earn CME/CNE This is a review of the major changes in the American Joint Committee on Cancer staging manual, eighth edition, for differentiated and anaplastic thyroid carcinoma. All patients younger than 55 years have stage I disease unless they have distant metastases, in which case, their disease is stage II. In patients aged 55 years or older, the presence of distant metastases confers stage IVB, while cases without distant metastases are further categorized based on the presence/absence of gross extrathyroidal extension, tumor size, and lymph node status. Patients aged 55 years or older whose tumor measures 4 cm or smaller (T1-T2) and is confined to the thyroid (N0, NX) have stage I disease, and those whose tumor measures greater than 4 cm and is confined to the thyroid (T3a) have stage II disease regardless of lymph node status. Patients aged 55 years or older whose tumor is confined to the thyroid and measures 4 cm or smaller (T1-T2) with any lymph node metastases present (N1a or N1b) have stage II disease. In patients who demonstrate gross extrathyroidal extension, the disease is considered stage II if only the strap muscles are grossly invaded (T3b); stage III if there is gross invasion of the subcutaneous tissue, larynx, trachea, esophagus, or recurrent laryngeal nerve (T4a); or stage IVA if there is gross invasion of the prevertebral fascia or tumor encasing the carotid artery or internal jugular vein (T4b). The same T definitions will be used for both differentiated and anaplastic thyroid cancer, but the basic premise of the anatomic stage groups will remain the same. CA Cancer J Clin 2018;68:55-63. © 2017 American Cancer Society.

Multi-regional sequencing reveals the genetic and immune heterogeneity of non-cancerous tissues in gastric cancer.

Gastric cancer (GC) is one of the most heterogeneous tumors. However, research on normal tissue adjacent to the tumor (NAT) is very limited. We performed multi-regional omics sequencing on 150 samples to assess the genetic basis and immune microenvironment in NAT and matched primary tumor or lymph node metastases. NATs demonstrated different mutated genes compared with GC, and NAT genomes underwent independent evolution with low variant allele frequency. Mutation profiles were predominated by aging and smoking-associated signatures in NAT instead of signatures associated with genetic instability. Although the immune microenvironment within NATs shows substantial intra-patient heterogeneity, the proportion of shared TCR clones among NATs is five times higher than that of tumor regions. These findings support the notion that subclonal expansion is not pronounced in NATs. We also demonstrated remarkable intra-patient heterogeneity of GCs and revealed heterogeneity of focal amplification of CD274 (encoding PD-L1) that leads to differential expression. Finally, we identified that monoclonal seeding is predominant in GC, which is followed by metastasis-to-metastasis dissemination in individual lymph nodes. These results provide novel insights into GC carcinogenesis. © 2024 The Pathological Society of Great Britain and Ireland.

KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival.

While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumor tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumor tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumor tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides insight into the complex OSCC tissue N-glycoproteome, thereby forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glycomarkers for OSCC.

Development and validation of a novel model to predict recurrence-free survival and melanoma-specific survival after sentinel lymph node biopsy in patients with melanoma: an international, retrospective, multicentre analysis.

BACKGROUND: The introduction of adjuvant systemic treatment for patients with high-risk melanomas necessitates accurate staging of disease. However, inconsistencies in outcomes exist between disease stages as defined by the American Joint Committee on Cancer (8th edition). We aimed to develop a tool to predict patient-specific outcomes in people with melanoma rather than grouping patients according to disease stage. METHODS: Patients older than 13 years with confirmed primary melanoma who underwent sentinel lymph node biopsy (SLNB) between Oct 29, 1997, and Nov 11, 2013, at four European melanoma centres (based in Berlin, Germany; Amsterdam and Rotterdam, the Netherlands; and Warsaw, Poland) were included in the development cohort. Potential predictors of recurrence-free and melanoma-specific survival assessed were sex, age, presence of ulceration, primary tumour location, histological subtype, Breslow thickness, sentinel node status, number of sentinel nodes removed, maximum diameter of the largest sentinel node metastasis, and Dewar classification. A prognostic model and nomogram were developed to predict 5-year recurrence-free survival on a continuous scale in patients with stage pT1b or higher melanomas. This model was also calibrated to predict melanoma-specific survival. Model performance was assessed by discrimination (area under the time-dependent receiver operating characteristics curve [AUC]) and calibration. External validation was done in a cohort of patients with primary melanomas who underwent SLNB between Jan 30, 1997, and Dec 12, 2013, at the Melanoma Institute Australia (Sydney, NSW, Australia). FINDINGS: The development cohort consisted of 4071 patients, of whom 2075 (51%) were female and 1996 (49%) were male. 889 (22%) had sentinel node-positive disease and 3182 (78%) had sentinel node-negative disease. The validation cohort comprised 4822 patients, of whom 1965 (41%) were female and 2857 (59%) were male. 891 (18%) had sentinel node-positive disease and 3931 (82%) had sentinel node-negative disease. Median follow-up was 4·8 years (IQR 2·3-7·8) in the development cohort and 5·0 years (2·2-8·9) in the validation cohort. In the development cohort, 5-year recurrence-free survival was 73·5% (95% CI 72·0-75·1) and 5-year melanoma-specific survival was 86·5% (85·3-87·8). In the validation cohort, the corresponding estimates were 66·1% (64·6-67·7) and 83·3% (82·0-84·6), respectively. The final model contained six prognostic factors: sentinel node status, Breslow thickness, presence of ulceration, age at SLNB, primary tumour location, and maximum diameter of the largest sentinel node metastasis. In the development cohort, for the model's prediction of recurrence-free survival, the AUC was 0·80 (95% CI 0·78-0·81); for prediction of melanoma-specific survival, the AUC was 0·81 (0·79-0·84). External validation showed good calibration for both outcomes, with AUCs of 0·73 (0·71-0·75) and 0·76 (0·74-0·78), respectively. INTERPRETATION: Our prediction model and nomogram accurately predicted patient-specific risk probabilities for 5-year recurrence-free and melanoma-specific survival. These tools could have important implications for clinical decision making when considering adjuvant treatments in patients with high-risk melanomas. FUNDING: Erasmus Medical Centre Cancer Institute.

Completion axillary lymph node dissection for the identification of pN2-3 status as an indication for adjuvant CDK4/6 inhibitor treatment: a post-hoc analysis of the randomised, phase 3 SENOMAC trial.

BACKGROUND: In luminal breast cancer, adjuvant CDK4/6 inhibitors (eg, abemaciclib) improve invasive disease-free survival. In patients with T1-2, grade 1-2 tumours, and one or two sentinel lymph node metastases, completion axillary lymph node dissection (cALND) is the only prognostic tool available that can reveal four or more nodal metastases (pN2-3), which is the only indication for adjuvant abemaciclib in this setting. However, this technique can lead to substantial arm morbidity in patients. We aimed to pragmatically describe the potential benefit and harm of this strategy on the individual patient level in patients from the ongoing SENOMAC trial. METHODS: In the randomised, phase 3, SENOMAC trial, patients aged 18 years or older, of any performance status, with clinically node-negative T1-T3 breast cancer and one or two sentinel node macrometastases from 67 sites in five European countries (Denmark, Germany, Greece, Italy, and Sweden) were randomly assigned (1:1), via permutated block randomisation (random block size of 2 and 4) stratified by country, to either cALND or its omission (ie, they had a sentinel lymph node biopsy only). The primary outcome is overall survival, which is yet to be reported. In this post-hoc analysis, patients from the SENOMAC per-protocol population, with luminal oestrogen-receptor positive, HER2-negative, T1-2, histological grade 1-2 breast cancer, with tumour size of 5 cm or smaller were selected to match the characteristics of cohort 1 of the monarchE trial who would only have an indication for adjuvant abemaciclib if found to have 4 or more nodal metastases. The primary study objective was to determine the number of patients who developed patient-reported severe or very severe impairment of physical arm function after cALND (as measured by the Lymphedema Functioning, Disability, and Health [Lymph-ICF] Questionnaire) 1 year after surgery to avoid one invasive disease-free survival event at 5 years with 2 years of adjuvant abemaciclib, using invasive disease-free survival event data from cohort 1 of the monarchE trial. The SENOMAC trial is registered with ClincialTrials.gov, NCT02240472, and is closed to accrual and ongoing. FINDINGS: Between Jan 31, 2015, and Dec 31, 2021, 2766 patients were enrolled in SENOMAC and randomly assigned to cALND (n=1384) or sentinel node biopsy only (n=1382), of whom 2540 were included in the per-protocol population. 1705 (67%) of 2540 patients met this post-hoc study's eligibility criteria, of whom 802 (47%) had a cALND and 903 (53%) had a sentinel lymph node biopsy only. Median age at randomisation was 62 years (IQR 52-71), 1699 (>99%) of 1705 patients were female, and six (<1%) were male. Among 1342 patients who responded to questionnaires, after a median follow-up of 45·2 months (IQR 25·6-59·8; data cutoff Nov 17, 2023), patient-reported severe or very severe impairment of physical arm function was reported in 84 (13%) of 634 patients who had cALND versus 30 (4%) of 708 who had sentinel lymph node biopsy only (χ2 test p<0·0001). To avoid one invasive disease-free survival event at 5 years with adjuvant abemaciclib, cALND would need to be performed in 104 patients, and would result in nine patients having severe or very severe impairment of physical arm function 1 year after surgery. INTERPRETATION: As a method to potentially identify an indication for abemaciclib, and subsequently avoid invasive disease-free survival events at 5 years with 2 years of adjuvant abemaciclib, cALND carries a substantial risk of severe or very severe arm morbidity and so cALND should be discouraged for this purpose. FUNDING: Swedish Research Council, the Swedish Cancer Society, the Nordic Cancer Union, and the Swedish Breast Cancer Association.