Miller, Jeffrey S.

Abstract Natural killer (NK) cells represent key effectors of antitumor immunity, yet emerging evidence highlights populations with distinct roles in cancer. Despite such expanded diversity within the NK cell repertoire, we lack an understanding of how this heterogeneity affects immune responses and downstream clinical outcomes. Using single-cell RNA sequencing, we systematically profiled NK cells across cancer and uncovered a dichotomous phenotypic and functional landscape of tumor-infiltrating NK cells shaped by opposing intrinsic signaling programs that drive the expression of IFNG or TGFB1. These divergent programs are associated with distinct transcription factor circuits that integrate cues within the tumor microenvironment and skew NK cells toward proinflammatory or suppressive functions. We found that the capacity for NK cells to engage in either functional direction is intrinsically linked to their phenotypic identity. Canonical NK cells recruited from circulation predominantly directed suppressive TGFB1 signals toward effector CD8+ T cells in tumors. Of note, these subsets exhibited higher TGFB1 expression than intratumoral myeloid cells across tumor types. In contrast, a tissue-resident (TR) adaptive subset exhibited exclusively proinflammatory IFNG-driven profiles and was associated with prolonged survival in both primary and metastatic tumor settings. Moreover, these TR adaptive NK cells, but not other subsets, were linked to response to immune checkpoint blockade. Collectively, our study reveals a previously unrecognized regulatory axis in NK cells that shapes NK cell diversity and augments broader antitumor immune responses.

Abstract Front-line pharmaceutical interventions for treating acute graft-versus-host disease (GVHD) are not uniformly effective and have toxic side effects. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent in vitro and in vivo immunosuppressive functions. Clinical translation of in vitro–generated MDSCs has been limited because of requirements for multiple, high infusion doses, the relatively low yield from peripheral blood–sourced MDSCs (PB-MDSCs), and inconsistent product quality. To circumvent these obstacles, we developed a methodology to generate MDSCs using human induced pluripotent stem cell (iPSC)–derived CD34+ cells. Compared with PB-MDSCs, iPSC-derived MDSCs (iMDSCs) shared similar morphology, phenotype, and suppressive function. We found that the CD14+ iMDSC subset possessed the highest suppressor function. In previous studies, we reported that MDSCs transferred into mice with GVHD lost suppressor function because of inflammasome activation and immature myeloid cell maturation. In striking contrast to human PB-MDSCs, we show herein that iMDSCs retained 95% of suppressor function in vitro despite exposure to lipopolysaccharide (LPS) plus adenosine triphosphate (ATP), which are stimuli that activate the inflammasome via danger-associated molecular patterns released during early posttransplant conditioning and GVHD-induced injury. In an in vivo xenogenic GVHD model with PB mononuclear cells, iMDSCs significantly increased recipient survival without loss of antileukemia effects. iMDSC RNA sequencing and gene knockdown studies revealed that the maintenance of the purine metabolizing enzyme, phosphoglycerate dehydrogenase, during LPS plus ATP treatment, was linked to iMDSC inflammasome resistance. Taken together, these findings provide a platform for translating in vitro–generated, off-the-shelf iMDSCs into the clinic for suppressing a spectrum of adverse immune responses, including GVHD.

e14555 Background: Natural killer (NK) cells are innate lymphocytes that engage in direct cytotoxic responses against malignant cells and promote pro-inflammatory tumor microenvironments. While studies have predominantly focused on conventional NK cell subsets, recent findings uncovered adaptive and tissue-resident (TR) subsets with unique functional properties. The influence of distinct NK cell subsets on tumor immunobiology and clinical outcomes remains unknown. Methods: Single-cell RNA-sequencing (scRNA-seq) of primary, therapy-naïve tumors from 276 patients across 14 cancer types, as well as 39 patients treated with immune checkpoint blockade (ICB), was accessed through publicly available datasets. We employed systematic computational analyses to phenotype and examine intra-tumoral NK cells. Gene signatures for NK cell subsets were applied to bulk RNA-sequencing from 212,105 tumor samples to delineate their associations with clinical outcomes using the Caris CODEai database. Results: Six main subsets of NK cells were identified in solid tumors, including conventional CD56 dim and CD56 bright NK cells, alongside adaptive, TR adaptive, TR CD56 bright , and TR CD56 bright /adaptive subsets. Across cancer, TR adaptive NK cells emerged as the most prognostic subset, with high infiltration conferring significantly longer overall survival (HR[95% CI] = 0.68[0.66-0.69], p < 0.001) than conventional CD56 dim and CD56 bright subsets. This was also observed in cohorts of head and neck, non-small cell lung, and esophageal cancers, particularly those treated with ICB. Notably, elevated TR adaptive NK cell infiltration was more prognostic than high tumor mutational burden in these settings. This was most apparent in ICB-treated head and neck cancers (HR = 0.48[0.41-0.57], p < 0.001 vs HR = 0.90[0.76-1.04], p = 0.16). Additionally, TR adaptive NK cells were strongly correlated to the infiltration of CD8 + T cells and B cells in tumors. Underlying these observations, our scRNA-seq analyses demonstrated that TR adaptive NK cells exhibit the highest immunomodulatory activity of all subsets, expressing high levels of pro-inflammatory cytokines ( CCL3 , CXCL13 , and IFNG ) and low levels of immunosuppressive cytokines ( TGFB1 ). Mechanistically, this activity was linked to the upregulation of transcription factors associated with NK cell activation, function, and persistence and downregulation of genes related to stress response and exhaustion. Interestingly, scRNA-seq from paired pre- and post-ICB-treated tumors revealed a unique expansion of cytotoxic GZMB + TR adaptive NK cells after treatment. Conclusions: TR adaptive NK cells represent a novel subset of NK cells that may drive anti-tumor immune responses, particularly in the context of ICB treatment. Features of this unique population may be harnessed to design more effective NK cell-based therapies against solid tumors.

Abstract Background: Given the dramatic rise in incidence of colorectal cancer in young adults, there remain limited treatment options effective in metastatic disease. Although T cell-based therapies have been successful in treating highly antigenic cancers, like melanoma and lung, they have not yet been shown to induce consistent and durable tumor regression in common epithelial malignancies, including metastatic colorectal cancer (mCRC). CISH (Cytokine inducible SH2 containing protein) is a novel intracellular checkpoint target demonstrating PD-L1 ligand independent mechanisms of enhanced anti-cancer activity. Given that CISH is not currently tractable via antibody or small molecule drug modalities, we employed CRISPR for precise inhibition of CISH in tumor infiltrating lymphocytes (TILs). Here, we provide the first clinical report of safety and anti-tumor activity of CISH edited TILs in 12 patients with metastatic gastrointestinal cancers (NCT04426669), including a clinical complete response (CR) in a young-adult patient with immunotherapy-refractory mCRC. Methods: Tumors were surgically resected for TIL harvest, followed by a rapid expansion protocol and CRISPR/Cas9 knockout (KO) CISH. CISH KO, neoantigen-reactive TILs were expanded, then infused following non-myeloablative lymphocyte depleting (LD) chemotherapy (cyclophosphamide & fludarabine) followed by high-dose IL-2. To establish mechanistic attribution of the CR, we used iR-RepSeq+ for serial analysis of the TCR immune repertoire. Results: Our CRISPR engineering led to KO of CISH in T cells with high-efficiency (&gt;90%) without detectable off-target editing. We safely dosed patients at all five dose levels (range: 1.91e8 to 9.93e10 cells). No dose-limiting toxicities attributable to the CISH-KO TIL product were observed. Adverse events were consistent with established risks of LD chemotherapy, IL-2, or disease progression. A durable ongoing complete response (&gt;21 months) was achieved in a patient with microsatellite instability-high (MSI-H) mCRC refractory to anti-PD1/CTLA-4 combination therapy. Conclusions: We provide the first clinical report of CISH checkpoint targeting via genetically modified T cell therapy, with complete response in a patient with mCRC. Persistence and expansion of unique TCR clonotypes detected in neoantigen responsive TIL was temporally consistent with spikes in CISH edited alleles detected by NGS assay; in the patient with CR, four of the clonotypes exhibiting prolonged persistence greater than one-year post-infusion exhibited significantly reduced or undetectable expression of CISH compared to the total infused TIL population. Given these findings, further investigation of CISH checkpoint inhibition, via gene and cell therapy, and next-generation small molecule drugging modalities, is underway. Citation Format: Emil Lou, Modassir S. Choudhry, Timothy K. Starr, Timothy Folsom, Jason Bell, Blaine Rathmann, Anthony P. DeFeo, Ji Hyun Kim, Nicholas Slipek, Zhaohui Jin, Darin Sumstad, Christopher A. Klebanoff, Katherine Ladner, Akshat Sarkari, R Scott McIvor, Thomas A. Murray, Jeffrey Miller, Madhuri Rao, Eric Jensen, Jacob Ankeny, Mahmoud A. Khalifa, Anil Chauhan, Benjamin Spilseth, Ajay Dixit, Paolo P. Provenzano, Wenjing Pan, Daniel Weber, Miranda Byrne-Steele, Tom Henley, David H. McKenna, Matthew J. Johnson, Beau R. Webber, Branden S. Moriarity. First-in-human trial in patients with metastatic colorectal cancer using CRISPR-engineered tumor infiltrating lymphocytes in which the intracellular immune checkpoint CISH is inhibited [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT269.

Abstract B7-H3 (CD276) is a transmembrane glycoprotein of the B7 immune checkpoint superfamily that has emerged as a promising therapeutic target. To better understand the applicability of B7-H3–directed therapies, we analyzed 156,791 samples comprising 50 cancer types to interrogate the clinical, genomic, transcriptomic, and immunologic correlates of B7-H3 mRNA expression. DNA (592-gene/whole-exome) and RNA (whole-transcriptome) sequencing was performed from samples submitted to Caris Life Sciences. B7-H3 high versus low expression was based on top and bottom quartiles for each cancer type. Patients’ overall survival was determined from insurance claims data. Pathway analysis was performed using gene set enrichment analyses. Immune cell fractions were inferred using quanTIseq. B7-H3 is expressed across several human malignancies including prostate, pancreatic, ovarian, and lung cancers. High B7-H3 expression is associated with differences in overall survival, possibly indicating a prognostic role of B7-H3 for some cancers. When examining molecular features across all cancer types, we did not identify recurrent associations between B7-H3 expression and genetic alterations in TP53, RB1, and KRAS. However, we find consistent enrichment of epithelial-to-mesenchymal transition, Wnt, TGFβ, and Notch signaling pathways. In addition, tumors with high B7-H3 expression are associated with greater proportions of M1 macrophages, but lower fractions of CD8+ T cells. We have begun to define the genomic, transcriptomic, clinical, and immunologic features associated with B7-H3 expression in 50 cancer types. We report novel clinical and molecular features of B7-H3–high tumors which may inform how current B7-H3 therapeutics should be deployed and prioritized. Significance: B7-H3–targeting therapeutics have shown promising results in initial clinical trials. In this pan-cancer analysis of B7-H3 mRNA expression, we found that B7-H3 exhibits robust expression in many common cancer types. These results may inform further development of B7-H3–targeting therapeutics and may guide clinical decisions for patients with limited treatment options.

AbstractPatient‐derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non‐resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold‐standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Importance In patients with severe aortic valve stenosis at intermediate surgical risk, transcatheter aortic valve replacement (TAVR) with a self-expanding supra-annular valve was noninferior to surgery for all-cause mortality or disabling stroke at 2 years. Comparisons of longer-term clinical and hemodynamic outcomes in these patients are limited. Objective To report prespecified secondary 5-year outcomes from the Symptomatic Aortic Stenosis in Intermediate Risk Subjects Who Need Aortic Valve Replacement (SURTAVI) randomized clinical trial. Design, Setting, and Participants SURTAVI is a prospective randomized, unblinded clinical trial. Randomization was stratified by investigational site and need for revascularization determined by the local heart teams. Patients with severe aortic valve stenosis deemed to be at intermediate risk of 30-day surgical mortality were enrolled at 87 centers from June 19, 2012, to June 30, 2016, in Europe and North America. Analysis took place between August and October 2021. Intervention Patients were randomized to TAVR with a self-expanding, supra-annular transcatheter or a surgical bioprosthesis. Main Outcomes and Measures The prespecified secondary end points of death or disabling stroke and other adverse events and hemodynamic findings at 5 years. An independent clinical event committee adjudicated all serious adverse events and an independent echocardiographic core laboratory evaluated all echocardiograms at 5 years. Results A total of 1660 individuals underwent an attempted TAVR (n = 864) or surgical (n = 796) procedure. The mean (SD) age was 79.8 (6.2) years, 724 (43.6%) were female, and the mean (SD) Society of Thoracic Surgery Predicted Risk of Mortality score was 4.5% (1.6%). At 5 years, the rates of death or disabling stroke were similar (TAVR, 31.3% vs surgery, 30.8%; hazard ratio, 1.02 [95% CI, 0.85-1.22]; P = .85). Transprosthetic gradients remained lower (mean [SD], 8.6 [5.5] mm Hg vs 11.2 [6.0] mm Hg; P &amp;lt; .001) and aortic valve areas were higher (mean [SD], 2.2 [0.7] cm 2 vs 1.8 [0.6] cm 2 ; P &amp;lt; .001) with TAVR vs surgery. More patients had moderate/severe paravalvular leak with TAVR than surgery (11 [3.0%] vs 2 [0.7%]; risk difference, 2.37% [95% CI, 0.17%- 4.85%]; P = .05). New pacemaker implantation rates were higher for TAVR than surgery at 5 years (289 [39.1%] vs 94 [15.1%]; hazard ratio, 3.30 [95% CI, 2.61-4.17]; log-rank P &amp;lt; .001), as were valve reintervention rates (27 [3.5%] vs 11 [1.9%]; hazard ratio, 2.21 [95% CI, 1.10-4.45]; log-rank P = .02), although between 2 and 5 years only 6 patients who underwent TAVR and 7 who underwent surgery required a reintervention. Conclusions and Relevance Among intermediate-risk patients with symptomatic severe aortic stenosis, major clinical outcomes at 5 years were similar for TAVR and surgery. TAVR was associated with superior hemodynamic valve performance but also with more paravalvular leak and valve reinterventions.

Acute leukemia is a constellation of rapidly progressing diseases that affect a wide range of patients regardless of age or gender. Traditional treatment options for patients with acute leukemia include chemotherapy and hematopoietic cell transplantation. The advent of cancer immunotherapy has had a significant impact on acute leukemia treatment. Novel immunotherapeutic agents including antibody-drug conjugates, bispecific T cell engagers, and chimeric antigen receptor T cell therapies have efficacy and have recently been approved by the US Food and Drug Administration (FDA) for the treatment of patients with acute leukemia. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop a clinical practice guideline composed of consensus recommendations on immunotherapy for the treatment of acute lymphoblastic leukemia and acute myeloid leukemia.

Abstract Abstract 1733 Introduction: Much remains to be elucidated regarding the etiology of myelodysplastic syndrome (MDS) and why some cases progress to acute myeloid leukemia. Further, there are few data on how accurate case classification is within state cancer registries and the impact of the 2008 WHO MDS classification modifications on clinical practice. We are conducting a large population-based prospective study of MDS to investigate risk factors for initial disease as well as disease progression. Our study includes comprehensive central review of all cases. Initial findings are reported here. Methods: We began evaluation and follow-up of all newly diagnosed MDS cases in Minnesota between the ages of 20 and 85 years from April 1, 2010 onward. Our process includes rapid case identification by the Minnesota Cancer Surveillance System (MCSS), contact of individual patients after primary physician approval, and, for interested patients, participation in an extensive questionnaire with medical history review and optional saliva sample for DNA. In concert, all cases pass through central medical review including: 1) independent pathology report +/− slide review by two hematopathologists to confirm diagnosis and subclassification; 2) independent cytogenetic review by a cytogeneticist; and 3) medical chart review by an oncologist to identify pertinent clinical history and prospectively calculate the International Prognostic Scoring System (IPSS) and WHO-based prognostic scoring system (WPSS) scores. Annual follow-up is planned to monitor for disease progression and outcome. In cases of unknown cytogenetics, IPSS calculations are not completed. In cases of unknown cytogenetics or diagnosis of MDS-U, not currently stratified in the WPSS, WPSS calculations are not completed. Results: We describe the first 67 patients enrolled on this study that have completed central review. The majority of patients were male (n=44) and the median age of the cohort at diagnosis was 71 (range 41–85). Table 1 Conclusion: Ours is the first large prospective population-based study of MDS that includes central pathology, cytogenetic, and clinical review of newly diagnosed patients with prospective calculation of IPSS and WPSS and plans for long term follow-up. This study is expected to enroll approximately 700 MDS cases over a five year period. While early in investigation, our initial review highlights the variability in distinct WHO diagnosis between initial cancer registry (i.e., MCSS) designation and designation after thorough central review of all appropriate diagnostic components. Our data suggest the robustness of the WHO RA, RARS, and RCMD classifications, the possible over-utilization of MDS-U classification, and highlight the under-representation of therapy-related MDS identification with registry designation. The impact of discordant diagnoses between MCSS and central review on therapeutic strategies is uncertain, but warrants continued evaluation and underscores the importance of complete central review in evaluation of MDS cases. Supported by NIH R01 CA142714. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Much remains unknown regarding the etiology of MDS and why some cases progress to acute myeloid leukemia (AML). With MDS reportable as a distinct myeloid malignancy for the first time only in 2001, there are few data on state cancer registry case identification and classification accuracy. To grasp the true incidence and the scope of this heterogeneous clonal hematopoietic malignancy, the accuracy of such information is essential. In our third year of a large population-based prospective study of MDS to investigate risk factors for initial diagnosis and disease progression, we report our experience with MDS case identification and classification according to both our state cancer registry and our comprehensive central review process Methods We began our identification and follow-up of all newly diagnosed adult MDS cases in Minnesota in April 2010. Our process includes rapid case identification by the Minnesota Cancer Surveillance System (MCSS), contact of individual patients, and their participation in an extensive questionnaire and medical history review. In concert, all cases pass through central medical review including: 1) independent pathology review by two hematopathologists; 2) independent cytogenetic review by a cytogeneticist; 3) integrated central pathology-cytogenetic review; and 4) medical chart review by an oncologist to identify pertinent clinical history and treatments and prospectively calculate risk stratification scores (International Prognostic Scoring System (IPSS)). Annual follow-up monitors for disease progression and outcome. Results We describe the first 161 patients enrolled who have completed central review. Our data highlight the excellent concordance between MCSS and central review for the MDS subtypes RAEB (n=40/50, 80%), RARS (n=20/23, 87%), and RCMD (n=19/25, 76%). However, we noted poor concordance for the MDS subtypes RA (n=1/7 (14%) with 5 cases re-classified as RCMD) and MDS-U (n=3/24(13%) with 21 cases re-classified into other MDS subtypes). Notably, our integrated central review process identified and reclassified 12 additional cases of t-MDS when all data were considered. Our central review process noted an additional 30 cases classified as MDS by MCSS that we deemed ineligible after central review due to insufficient morphology (n=20) or reclassification to chronic myelomonocytic leukemia (CMML) (n=5), MDS/MPD (n=3), or AML (n=4). Conclusion Ours is the first large prospective population-based study of MDS that includes central pathology, cytogenetic, and clinical review of newly diagnosed patients with prospective risk stratification calculation and long term follow-up. Our review highlights the variability in the diagnosis and subclassification of MDS between our state cancer registry and after integrated central review, with as many as 80% of cases in certain MDS subtypes being re-classified, and an approximately 20% ineligibility rate. The high proportion of cases submitted in MCSS as MDS-U that were re-classifiable into specific MDS subtypes following central review suggests a possible misapplication of this MDS category in a way not intended by the 2008 WHO guidelines. The additional classification of t-MDS by our central review process suggests that this subtype is likely underestimated by cancer registries. Assessment of therapeutic implications will be important to determine if these variabilities are consequential. Disclosures: No relevant conflicts of interest to declare.