The study aims to investigate the efficacy of stereotactic body radiation therapy (SBRT) in melanoma patients with oligometastatic disease (OMD), and to assess the prognostic value of the European Society for Radiotherapy and Oncology (ESTRO) and European Organization for Research and Treatment of Cancer (EORTC) nomenclature for these patients. This is a single-center, retrospective study including all melanoma patients with OMD (n = 66) receiving SBRT between 2010 and 2023. Patients were categorized based on the timing of SBRT of OMD according to the ESTRO/EORTC classification. We analyzed local control, progression-free survival (PFS), overall survival (OS), safety, and prognostic factors. The median follow-up was 72.5 months. Patients were categorized at the timepoint of SBRT as having de novo (n = 20), repeat (n = 25), or induced (n = 21) OMD. The most common OMD subcategories were repeat oligorecurrence (33.3

Intratumor heterogeneity drives disease progression, but even though subclonal copy number variation (CNV) is a major contributor to this heterogeneity1, its impact on cell phenotype is not fully understood. Here, by applying high-quality joint whole genome sequencing and mRNA profiling (co-sequencing) in single cells to patient samples across solid tumors and leukemias, we have analyzed the in vivo transcriptional effect of subclonal CNVs within a tumor. We found that gene dosage is generally additive in low and moderate copy states, but that cancer-type-specific compensation is common, and core promoter elements are associated with reduced additivity. We find that different classes of subclonal CNV impose varying degrees of transcriptional constraints on a cell, with highly amplified megabase-size regions associated with a strong effect both in cis and trans, while arm level CNVs generally exert a milder effect. We also describe a previously unappreciated class of tumors with transient clonality, where every cell is genetically highly distinct. We find that transient clonality is common in ovarian cancer and soft tissue sarcoma, that it is preceded by a whole genome duplication event, and that gene dosage in these tumors affects transcript abundance to a similar degree as in cancer with stable subclones.

Background:Inflammatory plasma proteins have been associated with poorer clinical outcomes in melanoma patients treated with immune checkpoint inhibitors (ICIs). Furthermore, the plasma levels of thymidine kinase activity (TKa), an indicator of proliferation, have also been associated with adverse outcomes in several cancers. The aim of this study was to explore the relationship between TKa, systemic inflammation and survival in ICI-treated melanoma patients. Materials and methods:Fifty-eight patients with unresectable metastatic melanoma who received anti-programmed cell death protein 1 monotherapy were included in the study. Pretreatment plasma samples were analyzed for TKa levels (DiviTum® TKa assay; Biovica) and inflammatory proteins using the Olink® Target 96 Inflammation panel, and these biomarkers were correlated with patient survival. Results:A panel of five unfavorable biomarkers was selected, comprising TKa, C-X-C motif chemokine ligand 8, C-C motif chemokine ligand 3, hepatocyte growth factor and S100 calcium-binding protein A12. An increasing number of elevated biomarkers in the panel was associated with a progressively worse prognosis. In the univariate analysis, when patients with four to five unfavorable biomarkers were compared with those with none, the hazard ratio (HR) was 2.43 [95% confidence interval (CI) 0.91-6.44, P = 0.075] for progression-free survival (PFS) and 10.62 (95% CI 3.11-36.26, P < 0.001) for overall survival (OS). When adjusting for sex, age, stage and lactate dehydrogenase (LDH), the HR was 1.97 (95% CI 0.61-6.41, P = 0.26) for PFS and 11.03 (95% CI 2.53-48.12, P = 0.001) for OS. Conclusion:This multi-biomarker panel was independently associated with poor OS in ICI-treated melanoma patients. This finding suggests a prognostic value of the panel, indicating its potential relevance for stratifying patients by risk, although further validation in larger cohorts is warranted.

Metastatic melanoma is a lethal form of cancer. Immunotherapy is recommended for metastatic melanoma and known to elicit beneficial response even after disease progression per RECIST. On-treatment samples hold irreplaceable predictive value. We collected needle biopsies from the metastases of different body sites from the same patient that progressed upon PD-1 treatment. Leveraging Direct Nuclear Tagmentation and RNA sequencing (DNTR- seq), we conducted single-cell genomic analyses on melanoma metastases and jointly sequenced the transcriptomes of each single melanoma cell. After 3 months of PD-1 inhibitor treatment, single-cell DNA-seq analyses on melanoma metastases from different anatomic locations displayed distinct cancer subclones evidenced by their copy number aberrations. Joint transcriptomic analyses of the dominant genomic subclones from each body site showed converging phenotypes, under the selective pressure of immunotherapy. Our preliminary analyses of on-treatment needle biopsies shed light on the challenges of treating heterogeneous tumors and underscore the pressing need to identify an ideal denominator to reach disease control. Citation Format: Muyi Yang, Solrun Kolbeinsdottir, Katarina Hammarlund, Vitali Grozman, Martin Enge, Suzanne Egyhazi-Brage, Hildur Helgadottir. Sub-clonal copy number variations between on-treatment lesions in immunotherapy resistant melanoma patients [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A108.

The melanocortin-1-receptor (MC1R) has a key role in melanocyte pigmentation regulation. Certain MC1R germline genetic variants (R alleles) result in deficient melanin production and are associated with red hair, freckling, UV sensitivity, and melanoma susceptibility. We aimed to address whether inherited polymorphisms in MC1R impact the efficacy of immune checkpoint inhibitors (ICI) in patients with metastatic melanoma. Patients with advanced melanoma undergoing ICI treatment were genotyped for MC1R variants. The patients were grouped by their germline MC1R R variants (≥ 1 or 0) and followed for treatment response, progression-free survival (PFS) and overall survival (OS). Of the 103 patients included, 39 (37.9%) had at least one MC1R R allele (MC1R-R-carriers), whereas 64 patients did not harbor any R allele (MC1R-R-non-carriers). The hazard ratio (HR) for PFS in MC1R-R-carriers was 0.60, (95% CI 0.37-0.98, p = 0.043). The HR for OS was 0.63 (95% CI 0.37-1.08, p = 0.091). While MC1R is closely associated with melanoma susceptibility, its impact on ICI efficacy has not been explored previously. MC1R-R-carriers with metastatic melanoma had improved PFS when treated with ICIs. If validated in larger cohorts, MC1R genotyping may serve as a factor helping to predict response to ICIs in melanoma patients.

“Pro-senescence therapy”, which triggers both permanent cell cycle arrest and an immune response, is a controversial new strategy for cancer treatment. To assess this strategy in melanoma, we performed a high throughput microscopy-based senescence screen utilizing a panel of melanoma cell lines with different driver mutations and a collection of clinical and experimental drugs. We found that vemurafenib and trametinib, which inhibit BRAFV600E and MEK1/2, respectively, induced senescence in some but not all BRAF-mutant cell lines. In contrast, palbociclib, BKM-120 and crizotinib, which inhibit CDK4/6, PI3K, and MET/ALK/ROS1, respectively, triggered senescence in most cell lines, irrespective of BRAF/NRAS mutation status, and overcame intrinsic and acquired vemurafenib resistance. The combination of palbociclib and crizotinib synergized to further enhance the senescence response in all cell lines irrespective of BRAF/NRAS mutation status, increased the expression of SASP factors, such as IL-1α and β, and HLA class I and other markers for recognition by NK and T cells. Further, this combination caused a significant increase in CD8+ T cells and pro-inflammatory macrophages in the tumor microenvironment and a marked reduction of mouse melanoma tumor growth that was dependent on CD8+ T cells, suggesting increased immune surveillance. Our findings suggest that pro-senescence therapy based on concomitant inhibition of both CDK4/6 and MET/ALK/ROS1 could be developed further as an alternative treatment strategy for melanoma. Significance Pro-senescence therapy based on combined targeting of CDK4/6 with Palbociclib and MET/ALK/ROS1 with Crizotinib inhibits melanoma tumor growth through anti-tumor immune response activation, providing an alternative treatment strategy for malignant melanoma. ### Competing Interest Statement LGL is cofounder of MyCural Therapeutics AB and has ownership interests in this company. KGW is cofounder and shareholder of Aprea Therapeutics, a company that develops novel anticancer therapy including APR-246 (Eprenetapopt). KGW has previously obtained research funding and salary from Aprea Therapeutics. KGW is board member of MyCural Therapeutics AB. LBo is founder of T-Hope Nordics AB and has ownership interests in this company. Knut and Alice Wallenberg Foundation, KAW 2013.0093 Cancerfonden, 24 3901 Pj, 21 1817 Pj, 19 0561 Pj

The human blood proteome provides a holistic readout of health states through the assessment of thousands of circulating proteins. In this study, we present a pan-disease resource to enable the study of diverse disease phenotypes within a harmonized proteomics dataset. By profiling protein concentrations across 59 diseases and healthy cohorts, we identified proteins associated with age, sex, and body mass index, as well as disease-specific signatures. This study highlights shared and distinct protein patterns across conditions, demonstrating the power of a unified proteomics approach to uncover biological insights. The dataset, covering 8262 individuals and up to 5416 proteins, serves as an online resource for exploring disease-specific protein profiles and advancing precision medicine research.

Immune-related adverse events (IRAEs) during therapy with immune checkpoint-inhibitors (ICIs) are common, and its management sometimes requires glucocorticoids (GC). Predictors for development of IRAEs and data about the impact of GC on clinical outcome are missing. We evaluated the impact of GC to treat IRAEs on clinical outcome, and plasmatic inflammatory proteins as predictors for IRAEs.Patients with melanoma (n=98) treated with ICIs at Karolinska University Hospital were included. Clinical information and data regarding prescription of systemic GC were collected. Baseline plasma samples (n=57) were analyzed for expression of 92 inflammatory proteins.Forty-four patients developed at least one IRAE requiring systemic GC and the most common was hypocortisolemia (n=11). A median overall survival (mOS) of 72.8 months for patients developing IRAEs requiring GC, 17.7 months for those who did not, and 1.4 months for individuals receiving GC at baseline was observed in Kaplan-Meier curves (p = 0.001). In immortal time bias adjusted analysis patients receiving steroids to treat IRAE survived slightly longer, even though this time trend was not statistically significant. The mOS was 29 months for those treated with GC within 60 days after ICIs start and was not reached for patients receiving GC later. The number of ICIs cycles was higher in subjects receiving GC after 60 days (p=0.0053). Hypocortisolemia occurred mainly in males (10/11) and correlated with favorable outcome. Male patients with hypocortisolemia had lower expression of IL8, TGF-alfa and FGF5 and higher expression of DNER.GC may be used to treat IRAEs without major concern. However, GC early during ICIs may impact clinical outcome negatively. The prognostic value of hypocortisolemia and inflammation proteins as biomarkers should be further investigated.

<p>Supplementary Table S1 - PDF file 80K, Supplemental table containing additional authentication data</p>

<p>Legend for Supplemental Figures S1-S4</p>

<p>Melanoma cells induce MDSC-like cells through COX-2/PGE2 production.</p>

PGE2 receptor EP3 is responsible for the activation of STAT-3 signaling on MDSC-like cells.

Unsupervised HCL of an external dataset of breast cancer metastases (GSE14018) using the 358 "breast cancer liver metastasis-selective genes" derived from SAM analyses of metastases in the test dataset.

Supplemental table containing additional authentication data.

Tumor-educated MDSC-like cells are potent suppressors of autologous T cells.

<p>Supplementary Figure S4 - PDF file 136K, PGE2 receptor EP3 is responsible for the activation of STAT-3 signaling on MDSC-like cells</p>

<p>Supplementary Table S1 - PDF file 80K, Supplemental table containing additional authentication data</p>

A. Association between the 17-gene signature and IntClust subgroups. P-value is from anova test. B. Relapse free survival for the subset of patients in IntClust subgroups 3, 7 and 8, which are enriched for Luminal A tumors. 73% of tumors with high expression belonged to IntClust subgroup 3, while 41% of tumors with low expression were of subgroup 7 and 8. C. Associations between the 17 liver metastasis-selective genes and RFS and D. OS for all patients included in GOBO irrespective of ER status.

Supplementary Figure S3 - PDF file 73K, Melanoma cells induce MDSC-like cells through COX-2/PGE2 production