Stein, Mariam I.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown clinical successes in B cell hematological malignancies. Two B cell maturation antigen (BCMA)-directed CART cell therapies treating multiple myeloma (MM) were approved by FDA but most patients relapse within 1-2 years. Ex vivo-expanded antigen specific T cells as cancer treatment rely on TCR-mediated killing of tumors. We hypothesized that arming ex vivo enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions. First, we expanded mucin 1 (MUC1)-activated T cells from patients with MM through ex vivo stimulation by MUC1 peptides-loaded autologous PBMCs for 19 days. Then, we engineered MUC1-activated T cells with 4-1BB-co-stimulated BCMA CAR to generate MUC1 TCR+BCMA CAR+ T cells. Baseline T cells without MUC1 stimulation were engineered with BCMA CAR as control. Baseline T cells, MUC1 T cells, baseline BCMA CART cells, or MUC1 BCMA CART cells were cocultured with luc+ RPMI-8226, a MUC1+BCMA+ MM cell line, for 3 days. MUC1 BCMA CART cells showed stronger tumor killing than BCMA CART or MUC1 T cells in all 3 patients (p<0.0001). MUC1 BCMA CART showed improved proliferation than BCMA CART or MUC1 T cells (p=0.0765, p<0.0001, respectively) after a 5-day stimulation by RPMI-8226. To exclude possibility of metabolic alterations by 19-day PBMC incubation leading to better T cell functions in MUC1 BCMA CART, baseline and MUC1-stimulated T cells from 3 healthy donors were processed for the same assays. Day-19 BCMA CART cells showed inferior tumor killing compared to baseline BCMA CART in all 3 donors (p<0.0001). Lower T cell proliferation was also observed in day-19 BCMA CART (p=0.0838). Our data indicate that improved T cell functions in MUC1 BCMA CART were unlikely due to metabolic changes and required stimulation by tumor antigens prior to CAR engineering likely through MUC1 TCR. Additionally, MUC1 T cells, BCMA CART, or MUC1 BCMA CART cells from tested patients were stimulated by RPMI-8226 for bulk RNA sequencing. Both BCMA CART and MUC1 T cells had distinct transcriptomic features from MUC1 BCMA CART cells. Differential gene expression analysis showed upregulation of T cell function-associated genes (RYR2, ITGA1, GNLY, GZMA, etc.) in MUC1 BCMA CART vs BCMA CART. Enrichment of T cell function-associated pathways was also observed in MUC1 BCMA CART. Analysis between MUC1 T cell and MUC1 BCMA CART showed upregulation of T cell function-associated genes (CSF2, CRTAM, IFNG, GZMB, etc.) and enrichment of immune response pathways in MUC1 BCMA CART. Our findings suggest that equipping ex vivo-enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions in vitro. Importantly, this provides a rationale for a therapeutic regimen where patients receive ex vivo-enriched tumor-induced T cells followed by CAR engineering. Persistence of the engineered T cells is being investigated Citation Format: Kun Yun, R. Leo Sakemura, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Truc N. Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli M. Stewart, Jennifer M. Feigin, Grace E. DeFranco, Sophia Y. Goldberg, Dominic A. Skeele, Mariam Stein, Dara Missan, Latha Pathangey, Long Mai, Hong Xia, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Leif Bergsagel, Michael Gustafson, Sandra Gendler, Saad S. Kenderian. BCMA CAR engineering arms T cells with superior functions post ex vivo stimulation by MUC1 peptide-loaded autologous PBMCs [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 6103.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p< 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p< 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).

The MN1::ETV6 gene fusion resulting from t(12;22)(p13;q12) has been rarely reported in myeloid neoplasms. We describe a 69-year-old male with newly diagnosed acute myeloid leukemia (AML) with erythroid differentiation and t(12;22)(p13;q12) demonstrated by conventional chromosome studies. Subsequent fluorescence in situ hybridization studies demonstrated a balanced ETV6 gene rearrangement (at 12p13). To further characterize this translocation, whole-genome sequencing was performed which confirmed t(12;22) with breakpoints involving the MN1 and ETV6 genes. Herein, we describe our case and review the literature to summarize the clinical and laboratory findings in patients with this rare but recurrent MN1::ETV6 gene fusion observed in myeloid neoplasms. Importantly, this case expands the clinical spectrum associated with the MN1::ETV6 gene fusion to include AML with erythroid differentiation. Lastly, this case demonstrates the importance of moving toward more comprehensive molecular testing to fully characterize the driver events in neoplastic genomes.

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.