BACKGROUND:Patients with BRAFV600E (ie, Val600Glu)-mutated non-small-cell lung cancer (NSCLC) can be treated with BRAF and mitogen-activated protein kinase (MEK) inhibitors, or with immune checkpoint inhibitors (ICIs) with or without chemotherapy. We aimed to investigate which initial systemic treatment should be prioritised in this population. METHODS:In this retrospective cohort study conducted across 17 centres in the USA, Italy, France, and Brazil, clinicopathological data were collected from participants aged 18 years and older with stage IV, treatment-naive, metastatic BRAFV600E-mutated NSCLC and with an Eastern Cooperative Oncology Group performance status of 0-3, who started first-line treatment with ICIs with or without chemotherapy (PD-1 or PD-L1 inhibitors with or without platinum-based chemotherapy) or BRAF and MEK inhibitors (dabrafenib and trametinib or encorafenib and binimetinib) between Jan 2, 2015, and July 11, 2024. The primary endpoint was overall survival with first-line ICIs with or without chemotherapy versus with BRAF and MEK inhibitors. FINDINGS:284 participants were identified for this study, of whom 88 (31%) received ICIs with or without chemotherapy and 196 (69%) received BRAF and MEK inhibitors. The median age of participants was 68 years (IQR 61-74), and 148 (52%) participants were female and 136 (48%) male. Participants in the ICIs with or without chemotherapy group had a higher history of smoking (73 [83%] vs 118 [60%]; p=0·0002) and a higher PD-L1 expression (≥50% in 58 [66%] vs 76 [39%], 1-49% in 16 [18%] vs 67 [34%], and <1% in eight [9%] vs 31 [16%]; p=0·0003) than those in the BRAF and MEK inhibitor group. At a median follow-up time of 45·0 months (95% CI 39·0-55·7), ICIs with or without chemotherapy were associated with improved median overall survival compared with BRAF and MEK inhibitors (40·9 months [95% CI 33·3-not reached] vs 25·2 months [19·9-31·1]; hazard ratio [HR] 0·69 [0·49-0·98], p=0·039). In subgroup analyses, ICIs with or without chemotherapy, compared with BRAF and MEK inhibitors, were associated with longer median overall survival in participants with a history of smoking (HR 0·60 [0·40-0·90], p=0·013), with a PD-L1 tumour proportion score of ≥1% or higher (HR 0·66 [0·45-0·98], p=0·039), aged 70 years or older (HR 0·54 [0·31-0·94], p=0·029), with TP53 co-mutations (HR 0·46 [0·27-0·79], p=0·0048), and without brain metastases (HR 0·66 [0·45-0·99], p=0·045). With BRAF and MEK inhibitors, frequencies of adverse events of any grade and of grade 3 and higher were similar whether administered as first-line therapy or as second-line therapy following ICIs with or without chemotherapy. INTERPRETATION:First-line ICIs with or without chemotherapy were associated with improved overall survival compared with BRAF and MEK inhibitors in participants with metastatic BRAFV600E-mutated NSCLC, particularly among specific subpopulations. These findings, although suggesting potential clinical relevance, remain exploratory and require confirmation from prospective studies. FUNDING:NextGenerationEU.

Melanoma patients treated with anti-PD1 or anti-PD1/anti-CTLA4 present intrinsic or acquired resistance to these immune checkpoint blockade (ICB) treatments. However, the interactions between tumor-stromal-immune cells in the microenvironment (TME) underlying these resistances remain unclear. In this study, we used a cohort of melanoma patients (n = 61) treated with ICB, pre-treatment (n = 33) and post-progression (n = 28), for whole-exome sequencing, single-nucleus RNA sequencing and high-resolution spatial imaging. We developed a framework to analyze response and resistance (intrinsic and acquired) via tumor-intrinsic programs, immune features, and tumor-stromal-immune interactions. Patients with low immune infiltration exhibited enrichment of macrophages associated with hypoxia and angiogenesis phenotypes. Samples with high immune infiltrates displayed an enrichment in lymphocytes, particularly TCF7+ CD8+ T cells. Pre-treatment samples from patients experiencing durable clinical benefit are encriched in memory B cells and plasmablast-like B cells.The presence of Bcells and of follicular dendritic cells in non-lymph node biopsies support the presence of tertiary lymphoid structures within the TME. Lastly, the presence of a TIMD4-expressing macrophage subset correlates with lymphocyte infiltration, indicating its potential role in promoting anti-tumor immunity. Our work defines TME immune profile interaction with tumor-stromal cells associated with clinical outcomes in melanoma. NCI HTAN Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

INTRODUCTION:The PHAROS primary analysis revealed robust antitumor activity and acceptable safety with encorafenib plus binimetinib in patients with BRAF V600E-mutant metastatic NSCLC (mNSCLC). We report results after 18 months of additional follow-up. METHODS:In this ongoing open-label, single-arm, phase 2 study, patients with BRAF V600E-mutant mNSCLC (59 treatment-naive and 39 previously treated) received encorafenib 450 mg once daily and binimetinib 45 mg twice daily. Primary end point was objective response rate (ORR). Secondary end points included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS:At this data cutoff, median treatment duration with encorafenib plus binimetinib was 16.3 months in treatment-naive and 5.5 months in previously treated patients; minimum follow-up was approximately 32 and 22 months, respectively. In treatment-naive patients, the ORR was 75%, median DOR was 40.0 months, median PFS was 30.2 months, median OS was not estimable (95% confidence interval: 31.3-not estimable), and the 3-year OS probability was 53%. In previously treated patients, the ORR was 46%, median DOR was 16.7 months, median PFS was 9.3 months, median OS was 22.7 months, and the 3-year OS probability was 29%. Overall, the most frequent treatment-related adverse events were nausea (52%), diarrhea (44%), fatigue (33%), and vomiting (30%). Treatment-related adverse events led to dose reductions and permanent treatment discontinuations in 25 (26%) and 16 (16%) patients, respectively. CONCLUSIONS:With longer follow-up, encorafenib plus binimetinib showed durable and clinically meaningful antitumor activity, especially in treatment-naive patients, with a manageable safety profile in patients with BRAF V600E-mutant mNSCLC. CLINICAL TRIAL INFORMATION:ClinicalTrials.gov Identifier: NCT03915951.

PURPOSE:Among patients with advanced non-small cell lung cancer (NSCLC) who discontinue immune checkpoint inhibitors (ICI) because of immune-related adverse events (irAE), post-discontinuation clinical outcomes and factors associated with disease progression after discontinuation are largely unknown. EXPERIMENTAL DESIGN:Clinicopathologic data were abstracted from patients with advanced NSCLC who received ICI and discontinued treatment because of irAE. Factors associated with post-discontinuation progression-free survival (PFS) and post-discontinuation overall survival (OS) were evaluated. RESULTS:Of 2,794 patients, 10% (N = 271) discontinued ICI because of irAE, and the median duration of ICI treatment before discontinuation for irAE was 5.9 months (range, 0.03-73.5). A longer treatment duration before discontinuation for irAE was associated with improved post-discontinuation outcomes: for patients on ICI for <3 months (N = 89), 3 to 6 months (N = 49), and >6 months (N = 133) before discontinuing for irAE, the median post-discontinuation PFS was 6.2, 13.9, and 25.8 months (P < 0.001), respectively, and the median post-discontinuation OS was 21.7, 42.7, and 86.9 months (P < 0.001), respectively. At multivariable analyses, predictors of longer post-discontinuation PFS were PD-L1 ≥ 50%, complete response/partial response (CR/PR) to treatment, and treatment duration before discontinuation between 3 to 6 months and >6 months; predictors of longer post-discontinuation OS were nonsquamous histology, CR/PR, and treatment duration before discontinuation >6 months. The use of immunosuppressive agents for toxicity management did not affect post-discontinuation outcomes. CONCLUSIONS:A longer treatment duration before discontinuation, a best objective response of CR/PR, PD-L1 ≥50%, and nonsquamous histology may help clinicians identify patients who may experience long-term disease control after discontinuation of ICI for irAE.

Genomic profiling of cancers informs diagnostic and prognostic classification and aids in selection of targeted therapeutics. Targeted, next-generation sequencing of cancer-specific genes is clinically feasible and enables comprehensive somatic reporting; without a matched germline specimen, germline alterations can confound analyses of the somatic profile and generate uncertainty in interpretation. This work reports the validation and implementation of optional matched tumor/germline sequencing in a precision cancer medicine program. DNA from 63 patient samples was analyzed using OncoPanel, a hybrid capture-based sequencing assay of 461 genes. Three analytical pipelines were implemented: tumor only, matched tumor/germline, and germline only. For matched tumor/germline, germline alterations in 19 genes with actionable/therapeutic implications were rescued. Retrospective analysis of the first 1600 matched cases was done to determine the potential clinical utility of this approach. Limit of detection for point mutations/insertions and deletions was 3% allele fraction; reproducibility was >98%. Matched tumor/germline concordance across 938 somatic calls was 100%. The average tumor mutational burden (TMB) was approximately 4 mutations/Mb lower than tumor-only sequencing. TMB-high patients were accurately reclassified as TMB-low in 14% of cases. Twenty-five percent of validation cases (14% after launch) had a pathogenic or likely pathogenic germline variant conferring cancer susceptibility; 14% of validation cases (7% after launch) harbored a germline variant of therapeutic significance. Matched tumor/germline sequencing is more accurate than tumor-only sequencing, while still encompassing all genomic findings that inform targeted therapy selection.

INTRODUCTION:Mutations in STK11, KEAP1, and SMARCA4 predispose to inferior immune checkpoint inhibitor (ICI) efficacy in NSCLC, particularly among KRAS-mutant cases. Nevertheless, the frequency, clinicopathologic features, and clinical impact of deletions in these genes are poorly characterized. METHODS:Clinicopathologic correlates of STK11, KEAP1, and SMARCA4 deletion were analyzed in cases of nonsquamous NSCLC at Dana-Farber Cancer Institute (DFCI). mRNA and LKB1 protein levels were assessed using The Cancer Genome Atlas. Clinical outcomes were analyzed in patients who received ICI with or without chemotherapy at DFCI and Memorial Sloan Kettering Cancer Center. Analyses of each deletion excluded cases with mutations in that gene. RESULTS:Among the 3194 cases of nonsquamous NSCLC, 14.7% had STK11 deletion (STK11DEL), 13.5% KEAP1 deletion (KEAP1DEL), and 13.7% SMARCA4 deletion (SMARCA4DEL). These deletions correlated with lower programmed death-ligand 1 expression and higher disease stage, tumor mutational burden, and aneuploidy. STK11DEL, KEAP1DEL, and SMARCA4DEL each correlated with lower corresponding mRNA expression, and STK11DEL with lower LKB1 protein expression. Among 767 patients treated with chemoimmunotherapy, these deletions were associated with worse objective response rates (STK11 31% versus 45%, p = 0.005; KEAP1 33% versus 45%, p = 0.03; SMARCA4 29% versus 45%, p = 0.0007), progression-free survival (STK11 hazard ratio [HR] = 1.5, p = 0.0001; KEAP1 HR = 1.4, p = 0.002; SMARCA4 HR = 1.6, p < 0.0001), and overall survival (STK11 HR = 1.7, p < 0.0001; KEAP1 HR = 1.5, p = 0.003; SMARCA4 HR = 1.7, p < 0.0001). The effect of these deletions on chemoimmunotherapy outcomes was comparable to the effect of mutations in these genes. Among 1267 patients treated with ICI alone, these deletions did not impact outcomes in the Memorial Sloan Kettering Cancer Center cohort but were generally associated with worse outcomes in the DFCI cohort among KRAS-mutant cases. CONCLUSIONS:STK11, KEAP1, and SMARCA4 deletions correlate with distinct clinicopathologic features, reduced programmed death-ligand 1 expression, and poor chemoimmunotherapy efficacy in NSCLC.

ABSTRACT:Preclinical studies have shown that interleukin (IL)-1β blockade can modulate the tumor microenvironment (TME) to activate antitumor immunity and, in combination with immune checkpoint inhibitors (ICIs), prevent cancer growth. Our study investigates if immune biomarkers in the TME affect outcomes in patients with non–small cell lung cancer (NSCLC) treated with the IL-1β inhibitor canakinumab plus an ICI-based therapy and describes canakinumab effects on the TMEs in these patients. Exploratory analyses were conducted in two prospective trials evaluating canakinumab combined with pembrolizumab-based regimens in patients with NSCLC: CANOPY-1 (first-line setting) and CANOPY-N (neoadjuvant setting). Immunohistochemistry (IHC) and transcriptomic analyses were performed on baseline tumor samples from CANOPY-1, and IHC and multiplex immunofluorescence analyses were performed on baseline and posttreatment tumor samples from CANOPY-N. Associations with clinical outcomes were evaluated. In CANOPY-1, in patients with low levels of T-cell infiltration in the tumor, the addition of canakinumab to a pembrolizumab-based regimen was associated with progression-free and overall survival improvements. Low levels of T-cell infiltration were associated with an immunosuppressive gene expression phenotype, supporting the role of low T-cell infiltration as a surrogate of an overall immunosuppressive TME. In CANOPY-N, a reduction in immunosuppressive cells in the TME was observed following canakinumab and pembrolizumab treatment. Our exploratory biomarker analyses from the CANOPY-1 and CANOPY-N trials suggest that IL-1β blockade may shift the TME toward an immune-activated status and that patients with immunosuppressive TME features could benefit from the addition of canakinumab to an ICI-based treatment. SIGNIFICANCE:Patients with NSCLC with immunosuppressive tumor features and low T-cell infiltration derive less benefit from ICI-based treatment. Biomarker analyses presented here suggest that these patients may benefit from the addition of anti-IL-1β therapy to their treatment.

Background: Among patients with ER+/HER2- breast cancer (BC), IHC-defined HER2-low and HER2-0 tumors do not appear to significantly differ molecularly (Tarantino P. et al. Nat Comm 2023). However, this may be due to lack of sensitivity of IHC, which is currently used to select patients for T-DXd treatment, despite major diagnostic challenges. To better characterize the biologic correlates of HER2-low expression, we analyzed molecular profiles by quantitative ERBB2 mRNA expression and characterized the features of tumors harboring ERBB2del, for which resistance to T-DXd has been suggested. Methods: Genomic, transcriptomic and proteomic data from ER+/HER2- BCs were analyzed from the METABRIC, TCGA (early BC) and MSK MetTrospism (metastatic BC, MBC) databases. Genomic landscapes, gene expression and HER2 protein expression were compared between subgroups based on HER2 mRNA tertiles (ERBB2-low, -intermediate, -high), by HER2 IHC score (IHC 0, 1+, 2+/not amplified) and by ERBB2del. Further validation was conducted in two DFCI cohorts, including patients with HER2- early BC with tumors tested for OncotypeDX between 1/2018 - 12/2022 and patients with MBC with tumors that had undergone next-generation sequencing (NGS) between 7/2013 - 12/2020. Results: All ER+/HER2- BCs from the METABRIC (n=1298), TCGA (n=524) and early DFCI (n=971) cohorts exhibited some degree of ERBB2 mRNA expression. In both METABRIC and TCGA, the ERBB2 mRNA high group was enriched in pathways related to ER signaling (Hallmark estrogen response early), while the ERBB2 mRNA low group was enriched in genes involved in cell proliferation (Hallmark E2F targets, MYC targets) and immune response (Hallmark allograft rejection, TNF signaling via NF-kB, IFN-α response, IFN-γ response). At the genomic level, PIK3CA mutations were enriched in the ERBB2 mRNA-intermediate and -high groups (both p<0.01), whereas TP53 mutations were enriched in the ERBB2 mRNA-low subgroup (p<0.001). Notably, ERBB2del were common in the ERBB2 mRNA-low group of both METABRIC and TCGA (35% and 54%), but less frequent in the ERBB2 mRNA-intermediate and -high cohorts (METABRIC: 9% and 4%; TCGA: 14% and 5%). No significant difference in the distribution of PIK3CA, TP53 and ERBB2del was noted between HER2 IHC subgroups. Among tumors with ERBB2-low mRNA, >90% of ERBB2del tumors had a concurrent TP53 heterozygous deletion (TP53del), compared with only 25-36% of non-ERBB2del tumors (both on chromosome 17). ERBB2del tumors were found enriched in the expression of genes related to cell proliferation and immune response, while non-ERBB2del BCs were enriched in genes related to ER signaling. ERBB2del tumors were found scattered across HER2 IHC subgroups (30.7% of IHC 0, 23.9% of IHC 1+, 15.8% of IHC 2+), had lower ERBB2 mRNA and HER2 protein levels, lower ER signaling and higher expression of proliferation genes within each HER2 IHC category. In the DFCI cohort of ER+/HER2- MBC with clinical NGS (n=749), the prevalence of ERBB2del was 14.3%, with 87.9% of ERBB2del tumors having concurrent TP53del. A significant difference in overall survival (OS) was observed between ERBB2del vs. non-ERBB2del tumors (33.8 vs 47.8 months, p=0.037), irrespective of the presence of TP53del, whereas no difference in OS was observed by HER2 IHC score (p=0.26). Similarly, in the MSK MetTrospism cohort (n=883), patients with ER+/HER2- MBC and an ERBB2del had worse OS (16.4 vs 30.8 months, p<0.0001), while the presence of TP53del was not associated with OS (32.1 vs 34.2 months; p=0.62). Conclusions: ERBB2del are frequent events in ER+/HER2- BC that are characterized by low ERBB2 mRNA levels, and are associated with TP53del, higher proliferation and immunogenicity, lower ER signaling and HER2 protein expression, and decreased OS in the metastatic setting. Citation Format: Paolo Tarantino, Xintao Qiu, Rong Li, Albert Grinshpun, Hersh Gupta, Melissa E. Hughes, Gregory Kirkner, Lynette Sholl, Bruce E. Johnson, Matthew L. Meyerson, Andrew D. Cherniack, Yijia Jiang, Ningxuan Zhou, Nancy U. Lin, Henry W. Long, Sara M. Tolaney, Rinath M. Jeselsohn. Molecular characterization of estrogen receptor (ER)+/HER2- breast cancer unveils a biologically and clinically distinct entity with ERBB2 hemizygous deletion (ERBB2del) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-03-20.

The phase II PHAROS study previously showed that encorafenib plus binimetinib has antitumor activity in patients with BRAF V600E-mutant metastatic non-small cell lung cancer (mNSCLC). In PHAROS, 98 patients (59 treatment-naïve; 39 previously treated) received encorafenib 450 mg once daily and binimetinib 45 mg twice daily. We report updated results from data cutoff of March 14, 2025. The median duration of treatment with both encorafenib and binimetinib was 16.3 months in treatment-naïve and 5.5 months in previously treated patients. After median follow-up for overall survival (OS) of 52.3 months in treatment-naïve patients, mOS was 47.6 months (95% CI, 31.3 to not estimable); 4-year OS probability was 49% (95% CI, 35 to 62). After median follow-up for OS of 48.2 months in previously treated patients, mOS was 22.7 months (95% CI, 14.1 to 32.6); 4-year OS probability was 31% (95% CI, 16 to 47). In treatment-naïve and previously treated groups, 58% and 26% received ≥1 subsequent systemic anticancer treatment, respectively. Safety profile remained consistent with that in previous analyses. Although comparisons across trials should be done cautiously, to our knowledge, encorafenib plus binimetinib was associated with the longest mOS reported to date with targeted treatment in patients with treatment-naïve BRAF V600E-mutant mNSCLC.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have significantly improved the treatment of hormone receptor (HR+) positive breast cancer. Clinical trials have demonstrated that addition of CDK4/6i to endocrine therapy (ET) improves both progression-free and overall survival in patients with HR+ metastatic breast cancer (MBC). Nevertheless, their impact remains limited by the eventual development of resistance. While a number of resistance mechanisms have been identified using both preclinical model systems and translational interrogation of patient specimens, these are not apparent in all cases. As part of the Human Tumor Atlas Network, we integrated genomic (whole-exome sequencing; n=47), single-nucleus transcriptomic (snRNA-seq; n=56), and spatial expression profiling (MERFISH and multiplex immunofluorescence; n=22 and n=17, respectively) from 58 clinically annotated MBC biopsy specimens collected before or after treatment with CDK4/6i plus ET to dissect the roles of tumor-intrinsic programs and the tumor microenvironment (TME) in CDK4/6i resistance. Transcriptomic analysis of the tumor cell compartment with snRNA-seq not only extended prior findings including an association between expression of cell-cycle programs and CDK4/6i resistance, but importantly identified additional programs associated with response and resistance including ciliogenesis, immune evasion, and inflammation. Additionally, tumor-intrinsic gene programs related to epithelial-to-mesenchymal transition had a decreased activity in ESR1 mutant biopsies, a mutational feature of CDK4/6i-resistant MBC associated with upregulated estrogen receptor pathway activity. In the TME, CD8+ T cells were depleted in the CDK4/6i-resistant biopsies, both before and after treatment. In contrast, the CDK4/6i-sensitive biopsies displayed pro-inflammatory features including an increased number of CXCL9+/CXCL10+ macrophages and clustering of CD8+ T cells in the vicinity of the malignant cells spatially. Notably, MERFISH also demonstrated an association between spatial architecture and CDK4/6i response, with the malignant cells in the CDK4/6i-resistant biopsies dispersed spatially compared to the sensitive biopsies. In summary, to the best of our knowledge, this study represents the largest spatially-resolved single-cell MBC cohort with curated CDK4/6i response status established to date. Our findings offer insights into potential biomarkers for CDK4/6i response based on tumor-intrinsic and tumor-microenvironment mechanisms, as well as therapeutic opportunities. Junko Tsuji, Jorge Gómez Tejeda Zañudo, Timothy R. Blosser, Ha T. Vu, Danielle Firer, Nick Maus, Kathleen L. Pfaff, Billie A. Porter, Jason L. Weirather, Anne Carlisle, Allison M. Frangieh, Elliot Boblitt, Aaron R. Thorner, Karla E. Helvie, Isabella E. Kallassy, Laura K. DelloStritto, Ragnhild Laursen, François Aguet, Mendy Miller, Melissa E. Hughes, Nancy U. Lin, Sara M. Tolaney, Eliezer M. Van Allen, Samouil L. Farhi, Xiaowei Zhuang, Barbara Engelhardt, Scott J. Rodig, Alex K. Shalek, Aviv Regev, Bruce E. Johnson, Nikhil Wagle, Gad Getz, Daniel L. Abravanel. Spatially-resolved single-cell transcriptome landscape of response and resistance to CDK4/6 inhibitors in patients with hormone receptor-positive metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6605.

PURPOSE:Inflammatory breast cancer (IBC) is a rare and clinically distinct form of breast cancer associated with poor outcomes. The biological mechanisms driving IBC remain poorly understood, partly due to limited large-scale genomic studies that directly compare IBC with non-IBC cases. EXPERIMENTAL DESIGN:We conducted a retrospective analysis of 140 patients with IBC (68 primary tumors and 72 metastatic tumors) and 2,317 patients with non-IBC (700 primary tumors, 65 local recurrences, and 1,552 metastases). We compared clinicopathologic features, single-nucleotide variants, copy-number variants, tumor mutational burden, and exploratory survival outcomes between IBC and non-IBC tumors. RESULTS:The most frequent somatic alterations in IBC were detected in TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%), and GATA3 (8%). Multivariate logistic regression revealed a significant enrichment of TP53 single-nucleotide variants in IBC, particularly in HER2+ and hormone receptor-positive disease. Tumor mutational burden did not differ between IBC and non-IBC cases. In HER2+ disease, a pathway analysis revealed an enrichment of NOTCH pathway alterations. TP53, CCND1, and RB1 alterations were associated with poor outcomes in IBC. CONCLUSIONS:This study provides a comprehensive resource of somatic alterations in a large cohort of patients with metastatic IBC and non-IBC, highlighting genomic features associated with worse outcomes. Our findings reveal a significant enrichment of TP53 mutations, reinforcing its critical role in IBC pathogenesis. Few other distinct differences in IBC were observed, suggesting further investigations-beyond bulk sequencing of the somatic genome-are required to better understand the biology driving this aggressive disease.

Objectives Well-differentiated neuroendocrine tumors (NET) are highly vascular tumors characterized by their expression of vascular endothelial growth factor (VEGF). This trial investigated the activity of ramucirumab, a monoclonal antibody that targets VEGF receptor-2 (VEGFR-2) and inhibits activity of VEGF, in combination with somatostatin analog therapy in patients (pts) with advanced extra-pancreatic NET.Methods We conducted a single-arm phase II trial enrolling pts with advanced, progressive extra-pancreatic NET. Patients were treated with ramucirumab 8 mg/kg intravenously on days 1 and 15 of each 28-day cycle. The primary endpoint was progression-free survival (PFS). Secondary endpoints toxicity, radiographic and biochemical tumor response rate, and overall survival (OS).Results The trial enrolled 43 patients. Primary tumor sites included small intestine 20 (46%), lung 10 (23%), thymus 3 (7%), rectum 1(2%), kidney 1(2%), and unknown primary 8(18%). Median PFS was 14.2 months (95% CI, 9.0-25.6 months), and median OS was 24.9 months (95% CI, 20.7-43.1 months). Best response by RECIST 1.1: partial response 5% (95% CI, 0.6%-15.8%). Chromogranin A levels dropped by at least 50% in 10% of the 37 patients who had elevated levels at baseline. Most common all-grade adverse events included fatigue (84%) and hypertension (84%).Conclusion Ramucirumab demonstrated efficacy and safety in this single-arm phase II trial. These findings support the continued evaluation of angiogenesis inhibitors in the treatment of NET.Clinical trial registration NCT02795858.

Tumor protein p53 (TP53) is the most frequently mutated gene across many cancers and is associated with shorter overall survival in lung adenocarcinoma (LUAD). Here, to define how TP53 mutations affect the LUAD tumor microenvironment (TME), we constructed a multiomic cellular and spatial atlas of 23 treatment-naive human lung tumors. We found that TP53-mutant malignant cells lose alveolar identity and upregulate highly proliferative and entropic gene expression programs consistently across LUAD tumors from resectable clinical samples, genetically engineered mouse models, and cell lines harboring a wide spectrum of TP53 mutations. We further identified a multicellular tumor niche composed of SPP1+ macrophages and collagen-expressing fibroblasts that coincides with hypoxic, prometastatic expression programs in TP53-mutant tumors. Spatially correlated angiostatic and immune checkpoint interactions, including CD274–PDCD1 and PVR–TIGIT, are also enriched in TP53-mutant LUAD tumors and likely engender a more favorable response to checkpoint blockade therapy. Our systematic approach can be used to investigate genotype-associated TMEs in other cancers. Zhao et al. used single-cell and spatial multiomics data analysis of human and mouse lung adenocarcinoma tumors and cell lines to reveal TP53 mutation-associated changes in cancer cells and their microenvironment.

Background: Breast cancer (BC) patients diagnosed at young ages (≤40y) often present with more aggressive tumors than older patients, highlighting the need to define differences in tumor biology by age. Limited data exists on tumor genomics in young metastatic BC (MBC) patients. Methods: The Ending Metastatic Breast Cancer for Everyone (EMBRACE) study is a prospective cohort enrolling women with MBC who receive care at Dana-Farber Cancer Institute. Women in EMBRACE with targeted sequencing conducted via Oncopanel for at least one tumor sample (collected at metastatic or primary diagnosis), were included. Tumors were classified by molecular subtype [HR+/HER2- (classified as luminal A- or B-like, with luminal-B like defined by primary tumor grade=3 or metastatic tumor progesterone receptor staining <10%), HR+/HER2+, HR-/HER2+, and HR-/HER2-]. After filtering for germline SNPs, oncogenic and likely oncogenic variants were selected. Tumor mutational burden (TMB) and variant frequencies were compared by age at MBC diagnosis (≤40y, >40-55y, >55y) using Fisher’s exact test, overall and within tumor molecular subtypes. Odds of individual gene mutations within age groups were compared using multivariable logistic regression models, adjusting for molecular subtype, histology, race, TMB, de novo v. recurrent MBC, and primary v. metastatic sample type. Kaplan-Meier curves, stratified by age group, assessed overall survival (OS, measured from MBC diagnosis) by genetic mutation status. Multivariable Cox regression analysis, adjusting for MBC diagnosis age, tumor factors, and race, was used to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for OS by gene mutation. Results: Tumor samples from 2,363 patients (1,594 metastatic, 769 primary) tested between July 2013-December 2020 were included. Median age at MBC diagnosis was 54 years (range: 18-91y). Median disease-free interval was 2.4 years (range: 0-33y), and younger women more likely to have de novo MBC (≤40y: 37% v. >55y: 23%, p<0.001). High TMB (≥10) was more common in older women with recurrent MBC (>55y: 28% v. ≤40y: 13%, p<0.001), though TMB did not differ by age for those with de novo MBC. After adjusting for tumor factors and race, compared to those >55y, patients ≤40y at MBC diagnosis were two to three times more likely to harbor mutations in BRCA1 [Odds ratio (OR) (95% CI)=3.11 (1.36-7.10), p<0.001], BRCA2 [OR (95% CI)=2.46 (1.15-5.27), p<0.001], and ERBB2 [OR (95% CI)=2.68 (1.46-4.90), p<0.001]. Mutations in GATA3 and TP53 were also more frequent among younger patients [OR (95% CI) GATA3 =1.68 (1.06-2.67), p<0.05; TP53=1.83 (1.22-2.73), p<0.01]. Mutations in CDH1 and PIK3CA were more common in older individuals [OR (95% CI) CDH1=0.07 (0.02-0.30), p<0.001, PIK3CA=0.45 (0.30-0.68), p<0.001]. Differences in OS by age were not statistically significant for de novo MBC patients, though OS was worse for recurrent MBC patients ≤40y v. >55y [median OS (95% CI)=2.8 (2.4-3.4y) v. 3.6 (3.3-3.9y), p=0.04]. In multivariable Cox models, mutations in TP53, MYC, and AKT1 were associated with shorter OS [HR (95% CI) TP53=2.05 (1.79-2.34), MYC=1.26 (1.01-1.56), AKT1=1.42 (1.10-1.84)], while mutations in GATA3, ERBB2, and MAP3K1 were associated with longer OS [HR (95% CI) GATA3=0.83 (0.69-1.00), ERBB2=0.53 (0.41-0.69), MAP3K1=0.75 (0.57-0.99)]. TMB was not associated with OS. Mutation-specific associations with survival did not differ by age group at diagnosis. Conclusions: In EMBRACE, differences in mutational frequency of several genes were observed by age at MBC diagnosis among patients with recurrent MBC, most notably for HR+/HER2- patients. Lower OS among younger recurrent MBC patients may be driven by these differences, particularly by high frequency of TP53 mutations in this age group. Further investigation of these genes is warranted to elucidate pathways leading to metastasis and to improve survival for young MBC patients. Citation Format: Kristen Brantley, Ananya Kodali, Gregory J. Kirkner, Melissa E. Hughes, Yvonne Li, Janet Files, Anne-Marie Feeney, Ayesha Mohammed-Abreu, Romualdo Barroso Sousa, Brittany Bychkovsky, Tari King, Bruce E. Johnson, Lynette Sholl, Deborah Dillon, Sara M. Tolaney, Andrew Cherniack, Ann H. Partridge, Nancy U. Lin, Ana C. Garrido-Castro. Tumor genomics in young patients with metastatic breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-09-19.

Immune checkpoint inhibitor (ICI) therapies have markedly improved the prognosis for patients with stage III & IV metastatic melanoma by prolonging progression-free and overall survival rates. However, the variability in immune evasion and resistance mechanisms presents significant challenges to the clinical efficacy of ICIs. This project aims to define drivers of immunotherapy response and resistance by employing advanced genomic, single-cell mRNA analyses, and spatial profiling techniques on tissue biopsies from metastatic melanoma patients. In this study, we developed a framework to analyze response and resistance, both intrinsic and acquired, via immune features in the tumor microenvironment in a standardized, uniformly processed, and deeply clinically annotated cohort of metastatic melanoma patients (n=61) treated with ICB as part of the human tumor atlas network (HTAN) initiative. From the tumor samples, we conducted single-nucleus RNA sequencing, and for a subset of the samples high-resolution spatial imaging (including protein mIHC, CODEX, and transcriptomics MERFISH). Standardized processing and data pipelines allowed for integrating genomic, transcriptomic, and spatial features to elucidate characteristics and mechanisms in tumor microenvironment and their relationships with resistance. Studies of the pretreatment samples demonstrated that CD4 and CD8 T cells, particularly TCF7+ CD8 T cells, are significantly more prevalent in responders to immunotherapy. Conversely, macrophages, especially Angio-TAMs, show higher levels of enrichment in non-responders. Moreover, the presence of B cells and follicular dendritic cells in non-lymph node biopsies supports the presence of tertiary lymphoid structures within the TME. Three levels of immune enrichment were identified through spatial analysis, and samples with more immune enrichment tend to have better responses. We also identified 10 recurrent cellular neighborhoods (RCNs) and found that RCN2,4, and 7 with high lymphocyte infiltration are significantly more enriched in responders than non-responders. In addition, RCN4 is associated with immune infiltration while RCN7 is TLS-like. Our findings indicate that patients with low immune infiltration exhibited enrichment of macrophages associated with the hypoxia and angiogenesis phenotypes, while patients with high immune infiltrates displayed enrichment in lymphocytes, particularly TCF7+ CD8+ T cells, confirming previous findings and indicating a robust T cell-mediated immune response. This project integrates genomic, transcriptomic, and spatial features to elucidate shared tumor and microenvironmental states and their relationships with resistance, and guide more personalized and effective treatment strategies for metastatic melanoma. Xinyu Cui, Giuseppe Tarantino, Yiwen He, Priyanka Solanky, Kathleen Pfaff, Aaron R. Thorner, Tyler J. Aprati, Boyang Zhang, Timothy Blosser, Emily Robitschek, Jiajia Chen, Junko Tsuji, Elliot Boblitt, Allison Frangieh, Hannah M. Faulkner, Marta Holovatska, Aleigha Lawless, Michael Manos, Karla Helvie, Tatyana Sharova, Dennie Frederick, James L. Fahey, Diego Villamarin, Sachi Krishna, Chanell Mangum, Ajit J. Nirmal, Domenic Abbondanza, Cai McCann, Bruce Johnson, Alex K. Shalek, Eliezer Van Allen, Xiaowei Zhuang, Ryan Sullivan, Barbara E. Engelhardt, Samouil L. Farhi, Scott Rodig, F. Stephen Hodi, Genevieve M. Boland, David Liu. Dissecting tumor-immune microenvironment in response and resistance to immune checkpoint blockade in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4536.

PURPOSE:Retrospective studies have found associations between the number of intratumoral immune cells and patient outcomes for specific cancers treated with targeted therapies. However, the clinical value of routinely quantifying intratumoral immune biomarkers using a digital pathology platform in the pan-cancer setting within an active clinical laboratory has not been established. METHODS:We developed ImmunoProfile, a daily clinical workflow that integrates automated multiplex immunofluorescence tissue staining, digital slide imaging, and machine learning-assisted scoring to quantify intratumoral CD8+, PD-1+, CD8+PD-1+, and FOXP3+ immune cells and PD-L1 expression in formalin-fixed, paraffin-embedded tissue samples in a standardized and reproducible manner. We prospectively applied ImmunoProfile to biopsies collected from 2,023 unselected patients with cancer over a 3-year period in the clinical laboratory and correlated the results with patient survival. RESULTS:In the pan-cancer cohort, patients with high numbers of intratumoral CD8+ or PD-1+ cells in had significantly lower risks of death compared with those with low numbers (CD8+: high v low hazard ratio [HR], 0.62 [95% CI, 0.48 to 0.81], Wald P = .002; PD-1+: high v low HR, 0.65 [95% CI, 0.51 to 0.83]; P = .0009) after adjusting for risk factors, including cancer type. In subset analyses, patients with high numbers of intratumoral CD8+, PD-1+, and/or CD8+PD-1+ cells showed lower risks of death from non-small cell lung, colorectal, breast, esophagogastric, head and neck, pancreatic, and ovarian cancers after considering clinical risk factors, including American Joint Committee on Cancer stage, and despite varying therapies (all P < .05). CONCLUSION:Routinely quantifying intratumoral CD8+ and PD-1+ cells with a clinically validated digital pathology platform predicts patient survival across major cancer types, independent of clinical stage and despite diverse treatment regimens.

Understanding the biological mechanisms underlying differential responses to immunotherapy is critical for advancing treatment strategies in metastatic melanoma. We analyzed single-nucleus RNA sequencing data from 66 melanoma samples, including 18 with matched spatial transcriptomics data via MERFISH, across metastatic sites such as lymph nodes, colon, skin, lung, brain, and liver. Our study aimed to identify tumor-intrinsic transcriptional programs and their association with durable clinical benefit. We employed non-negative matrix factorization to delineate distinct tumor programs within tumor cells. To assess the robustness and biological relevance of these programs, we conducted hallmark pathway analyses and utilized spatial transcriptomics to examine their associations with biopsy sites, response, and immune infiltration. Comparisons with published datasets were performed to characterize and ensure the stability and applicability of these programs. We identified five distinct tumor programs with differential enrichment between responders and non-responders. Tumor programs enriched in responders demonstrated strong associations with interferon alpha-beta signaling and antigen presentation pathways. This program was also enriched across diverse lymphocyte populations. Via MERFISH analysis, we classified patients into three categories based on the spatial distribution of immune cells relative to tumor borders: immune-enriched, immune-excluded, and immune-desert. Notably, the program enriched in responders was prevalent in the immune-excluded samples, suggesting that robust signaling pathways can drive therapeutic responses even in the absence of high immune infiltration. In contrast, a mesenchymal-like tumor program was predominant in non-responders, characterized by elevated epithelial-mesenchymal transition activity and diverse tumor-associated macrophage infiltration. This program was particularly enriched in colon metastases. Our findings underscore the importance of interferon signaling in driving therapy response and the role of mesenchymal-like programs in resistance. The unique association of immune-excluded but therapy-responsive samples with interferon-related pathways raises intriguing questions about tumor-immune dynamics. By integrating single-cell and spatial transcriptomics, this study provides insights into tumor heterogeneity and the tumor microenvironment, offering avenues for targeted therapeutic interventions. Future research will focus on spatial interactions, the characterization of immune subpopulations within tumor programs, and the dynamics of cell-cell communication between tumor and immune cells, aiming to refine and advance personalized treatment strategies. Priyanka Solanky, Giuseppe Tarantino, Yiwen He, Xinyu Cui, Kathleen Pfaff, Aaron R. Thorner, Tyler J. Aprati, Boyang Zhang, Timothy Blosser, Jiajia Chen, Junko Tsuji, Elliot Boblitt, Allison Frangieh, Hannah Mae Faulkner, Marta M. Holovatska, Aleigha R. Lawless, Michael Manos, Emily Robitschek, Karla Helvie, Tatyana Sharova, Dennie Frederick, James Liam Fahey, Diego Villamarin, Sachi Krishna, Chanell Mangum, Ajit J. Nirmal, Domenic Abbondanza, Cai McCann, Bruce Johnson, Alex Shalek, Eliezer Van Allen, Xiaowei Zhuang, Ryan J. Sullivan, Barbara Engelhardt, Sami Farhi, Scott J. Rodig, Stephen Hodi, Genevieve Boland, David Liu. Dissecting tumor-intrinsic programs and immune dynamics driving response to immunotherapy in metastatic melanoma through non-negative matrix factorization decomposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6256.

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare type of breast cancer associated with a unique clinical presentation and overall poor outcomes, recognized as a distinct category by the AJCC staging system. The biological mechanisms driving the IBC phenotype are relatively undefined—partially due to a lack of comprehensive, large-scale genomic studies and limited clinical cohorts. Here, we report one of the largest, subtype-informed clinicogenomic characterizations of IBC to date. METHODS: A retrospective analysis of 2457 patients with metastatic breast cancer who underwent targeted tumor-only DNA-sequencing (OncoPanel, up to 447 cancer-associated genes) was performed at Dana-Farber Cancer Institute. Clinicopathologic, single nucleotide variant (SNV), copy number variant (CNV) and tumor mutational burden (TMB) comparisons were made between IBC and non-IBC cases. Median follow-up was 28.1 months. RESULTS: Our profiled cohort included 140 patients with IBC specimens (n = 68 primary tumors, 72 metastatic tumors) and 2317 patients with non-IBC specimens (n = 702 primaries, 65 local recurrences, 1550 metastases). Of these, 87.4% of patients were White, 4.7% Black, 3.6% Asian or Pacific Islander, and 4.3% other/unknown. Clinicopathologic differences between IBC and non-IBC cases were consistent with previous reports—including younger age at diagnosis of metastatic disease (51 vs 54 years, p = 0.04), and a higher proportion of grade 3, estrogen receptor-negative and HER2-positive tumors (p < 0.001). Among the hormone-receptor (HR)-positive subtype, IBC tumors showed a significant enrichment in Luminal B (LumB)-inferred disease (62.5% vs 39.8%, p < 0.005), defined as tumors with grade 3 or progesterone receptor staining < 10%. The most recurrent somatic alterations spanning all subtypes in IBC were TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%) and GATA3 (8%). A multivariate logistic regression analysis accounting for HR and HER2 status revealed a significant enrichment in TP53 SNVs in IBC vs non-IBC (OR 2.08 [95% CI 1.34-3.24], adjusted p-value 0.04). Frequency of TP53 alterations in IBC vs non-IBC cases was 85.1% vs 64.3% in HER2-positive and 50.0% vs 27.7% in HR-positive disease—with an enrichment of TP53 mutations in IBC LumB-inferred tumors vs non-LumB (64% vs 26%, p < 0.05). When comparing HR+ IBC LumB vs HR+ non-IBC LumB cases, TP53 mutations were again enriched (p < 0.05)—suggesting LumB-like histopathology is not the only driving feature of TP53 enrichment in HR+ IBC. TMB did not differ substantially between IBC and non-IBC and no other statistically significant enrichments were observed, including when grouping mutations into six canonical cancer pathways (cell cycle, Notch signaling, PI3K pathway, RTK/RAS signaling, TP53 pathway and WNT signaling). CONCLUSIONS: Taken together, this study provides a comprehensive landscape of somatic alterations in a large cohort of patients with metastatic IBC and non-IBC. Our data support a lack of major genomic differences other than enrichments in TP53 mutations and an associated LumB-like histopathology. These results both reinforce the importance of TP53 mutations in IBC biology and suggest additional analyses beyond somatic DNA-level changes are warranted. Future efforts with the DFCI IBC cohort will assess germline-somatic interactions, non-genomic or transcriptomic characterizations, and potential environmental influences to better understand the mechanisms driving this unique disease. Citation Format: Nolan Priedigkeit, Beth Harrison, Melissa Hughes, Robert Shue, Yvonne Li, Gregory Kirkner, Claire Remolano, Sarah Strauss, Janet Files, Anne-Marie Feeney, Ayesha Mohammed-Abreu, Ana Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Faina Nakhlis, Jennifer Bellon, Tari King, Bruce Johnson, Lynette Sholl, Deborah Dillon, Beth Overmoyer, Sara Tolaney, Andrew Cherniack, Nancy Lin, Filipa Lynce. Comprehensive clinicogenomic characterization of inflammatory 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 PO1-14-10.

First round of screening of newly developed monoclonal antibody and dimeric nanobody reagent pairs in patient plasma.

SUMMARY Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org .