SpaceMarker result for all virtual patients

BACKGROUND:Accumulating evidence has demonstrated that distinct tumour-promoting and tumour-restraining cancer-associated fibroblast (CAF) subtypes coexist in pancreatic ductal adenocarcinoma. OBJECTIVE:To develop targeted CAF therapeutic strategies by reprogramming tumour-promoting CAF subtypes. DESIGN:We leveraged multiomics technologies to systematically identify and characterise CAF subtypes transcriptionally, epigenetically and spatially and correlate them with clinicopathological features. RESULTS:We found that complement-secreting CAFs (csCAFs), initially identified by our group and inflammatory CAFs (iCAFs) share significant overlap in their transcriptional profiles and chromatin accessibility. iCAFs specifically express transcription factors from the heme and oxidative homeostasis pathway and the activator protein 1 family, which are both involved in cellular response to oxidative stress. Notably, the composition of csCAFs among all CAFs declined during pancreatic carcinogenesis, while trajectory analysis showed that csCAFs could potentially differentiate into iCAFs. Spatially resolved analysis indicated that tumour regions with a higher csCAF composition were associated with lower levels of TGF-β ligands, fewer M2 tumour-associated macrophages and increased levels of lipid mediators. Additionally, we identified a spatially defined CXCL12-CXCR4 ligand-receptor interaction between csCAFs and T cells, but in distinct patterns between different metastatic organs. Patients with a higher composition of csCAFs have significantly longer overall survival and recurrence-free survival through multiplex immunohistochemistry and bulk RNA-seq deconvolution. CONCLUSION:Our study demonstrates that csCAFs may represent an early-stage iCAF subtype and suggests a promising strategy for reprogramming iCAFs into csCAFs.

CyTOF profiling of T cells in the peripheral blood

Multiplexed protein imaging enables spatial analysis of complex tissues, but detecting proteins expressed at low levels remains challenging, particularly in widely available formalin-fixed, paraffin-embedded (FFPE) specimens. Many biologically important regulators-including senescence markers, transcription factors, and secreted proteins-are therefore difficult to study in situ using existing high-plex methods. Here we show that integrable Co-detection of Low-Abundant Proteins (iCLAP) enables sensitive and highly multiplexed protein detection within the same FFPE tissue section. iCLAP combines iterative signal amplification with efficient fluorophore inactivation, enabling repeated staining of the same tissue section and seamless integration with established multiplex imaging platforms to achieve profiling of more than 40 markers. Application of iCLAP to human pancreatic tissues revealed spatially distinct senescence-associated protein patterns across tissue compartments. Together, iCLAP expands the analytical capabilities of FFPE tissues, enabling high-sensitivity, high-dimensional spatial proteomic studies of complex biological processes.

Immune checkpoint inhibitors (ICIs) have limited activity in mismatch repair proficient or microsatellite stable (MMRp/MSS) colorectal cancer (CRC). KRAS mutations, present in approximately 40% of these cancers, can generate neoantigens that are targets for therapeutic vaccines. In this single-arm, phase I study (NCT04117087), we evaluated mKRAS-VAX, a pooled mutant KRAS (mKRAS) peptide vaccine targeting six KRAS mutations with nivolumab and ipilimumab in 13 patients with pretreated metastatic MMRp/MSS CRC. Both primary endpoints of safety and immunogenicity (within 17 weeks post-vaccination) were met. Secondary endpoints included treatment efficacy defined by RECIST v1.1 criteria. All adverse events attributed to mKRAS-VAX were grade 1 or 2, and the addition of mKRAS-VAX did not increase the frequency of severe immune-related adverse events beyond the expected profile of dual ICIs alone. mKRAS-VAX elicited an increase in tumor-specific mKRAS-reactive T-cells in 8/12 biomarker-evaluable patients (75%) by direct ex vivo IFNγ ELISpot and in 12 patients (100%) following in vitro expansion. Our findings support further development of mKRAS vaccines with ICIs for advanced MMRp/MSS CRC.

e16301 Background: Surgical resection is the mainstay for localized, resectable biliary tract cancers (BTCs). Although genomic clusters have been associated with outcomes in BTCs, data linking specific molecular subtypes to post-surgical outcomes remain limited. We aimed to evaluate the prognostic role of tumor genetic signatures in patients with BTC undergoing curative-intent surgery. Methods: We retrospectively analyzed Liver Multi-Disciplinary Clinic (LMDC) patients who underwent curative-intent surgery between 2015 and 2025 with pathology proven BTC. We included patients with detailed pathological records and next generation sequencing (NGS) results. Our endpoints of disease-free survival (DFS) and overall survival (OS) were defined as the time from surgery to their first documented disease recurrence and death from any cause, respectively. Results: There were 97 patients (52.6% male, 47.4% female) in our cohort with a mean age of 65.1 years. Most patients had AJCC Stage II (28.8%) or III disease (27.8%). Median DFS was 20.1 months (CI 16.0–28.7) and median OS was 40.4 months (CI 37.3-52.5). Recurrence occurred in 64.2% of patients, and the median survival post-recurrence was 15.4 months. In gene-level analyses, alterations in SMAD4 and BRAF were significantly associated with shorter DFS (SMAD4: HR 4.2, CI 1.61-11.00, p 0.003; BRAF HR 3.89, CI 1.17-12.92, p 0.03). Mutations in SMAD4 and NTRK1/2/3 were also associated with worse OS (SMAD4: HR 3.53, CI 1.22-10.19, p 0.02; NTRK1/2/3: HR 7.72, CI 2.23 - 26.70, p 0.001), while mutations in FGFR2 significantly improved OS (HR 0.22, CI 0.05-0.92, p 0.03). Subsequently, cluster-based analysis was performed to prevent distortion from low-frequency alterations. Cluster 1 (TP53/KRAS/ATM) and cluster 2 (CDKN2A/2B) were not associated with OS or DFS. In contrast, cluster 3 (ARID1A/PBRM1/IDH1) alterations were associated with decreased OS (HR 2.36, CI 1.23-4.56, p 0.01). Cluster 4 (FGFR2/BAP1) mutations were associated with improved OS (HR 0.33, CI 0.12, 0.92, p 0.03) relative to patients without these alterations. Conclusions: In the post-surgical setting, patients harboring mutations in chromatin remodeling pathways (ARID1A/PBRM1/IDH1), as well as SMAD4 and NTRK1/2/3, experienced worse outcomes, whereas those with BAP1 and FGFR2 alterations demonstrate improved outcomes. Although limited by sample size, our study lays out the foundation for future investigations evaluating the impact of molecular profiling in BTC patients undergoing surgical resection.

Supplemental mathematical equations of the spQSP model

In this Phase II study (NCT02648282) of locally advanced pancreatic cancer (LAPC), 58 patients were enrolled and intended for an immunotherapy including GM-CSF-secreting allogeneic pancreatic cancer vaccine (GVAX) and pembrolizumab and stereotactic body radiation therapy (SBRT) following standard chemotherapy. Fifty-four evaluable patients received two cycles of immunotherapy and SBRT and reassessed for resectability. After resection or biopsy, patients received continued immunotherapy for two years. At a median follow-up of 19.6 months, the median distant metastasis free survival (DMFS), the primary endpoint, for all evaluable patients was 9.8 months. Secondary endpoint analysis shows that, of these, 35 patients (64.8%) were deemed potentially resectable, and 24 underwent R0/R1 resections. Resected patients had a majority of R0 resections (91.7%). The overall resection rate was 44.4%. Patients who underwent resection had a median DMFS of 20.3 months. Median overall survival (OS), a secondary endpoint, for all evaluable patients from the start of the immunotherapy was 21.8 months, with resected patients reaching 29.7 months and unresected patients at 12.1 months. From diagnosis, median OS was 28.2 months, with resected patients achieving 36.7 months, while unresected patients had 19.7 months. In conclusion, the combination of GVAX, pembrolizumab, and SBRT showed promising efficacy in LAPC patients with favorable survival outcomes, especially those who underwent R0/R1 resections. However, patients with unresectable disease still maintained significantly worse DMFS and OS. The regimen demonstrated a manageable safety profile.

Antibody-mediated rejection (AMR) presents a rare but complex challenge following liver transplantation, characterized by histopathologic features lacking specificity. The 2016 Banff Working Group criteria aimed to standardize AMR diagnosis, focusing on histologic findings and C4d staining interpretation. Our retrospective analysis of 463 liver transplant recipients between 2017–2023 identified 13 donor specific antibody (DSA)-positive cases (2.8

Abstract Background: Fibrolamellar carcinoma (FLC) is a rare and aggressive primary liver cancer that primarily affects adolescents and young adults. It is biologically distinct from other forms of primary liver cancer, such as hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). There are no standard or approved systemic therapies for advanced FLC, and most patients present with unresectable disease or experience recurrence after surgery. These factors underscore the need for new therapeutic approaches. Antibody-drug conjugates (ADCs) have shown efficacy in several solid tumors by delivering cytotoxic payloads to tumor-associated surface antigens with improved specificity, but their utility in FLC is not known. To evaluate the relevance of ADC-based therapies in FLC, we assessed the expression of five clinically actionable ADC targets (NECTIN4, TROP2, CLDN18.2, HER2, and B7-H3) in an expanded cohort of FLC specimens. Methods: Archival FFPE samples were obtained from consented patients through the Johns Hopkins Liver Cancer Tissue Bank and the Fibrolamellar Cancer Foundation, including FLC tumors (n=58; 38 primary and 20 metastatic lesions). Immunohistochemistry (IHC) was performed for all five markers. Expression patterns of interest in FLC were compared with control cohorts of HCC (n=10) and CCA (n=10). Three pathologists, blinded to clinical data, independently scored staining using both a 0 to 3 plus ordinal IHC scale and a semi-quantitative H-score. Results: Among the five ADC targets evaluated, only B7-H3 and HER2 showed detectable staining in FLC. NECTIN4, TROP2, and CLDN18.2 were uniformly negative, scored as 0+ in all tumors, and had H-scores of 0. B7-H3 demonstrated the strongest expression. 3+ staining was observed in 20 of 58 tumors (34.5%). Tumor-cell H-scores ranged from 10 to 240, with a mean of 132. B7-H3 was also expressed in the tumor microenvironment, with prominent staining in cancer-associated stroma and fibroblasts. Stromal H-scores ranged from 40 to 300, with a mean of 199, often exceeding tumor-cell expression. HER2 showed detectable 3+ staining in 12 of 58 tumors (21.7%), with H-scores ranging from 2 to 270 and a mean of 70. One patient in this cohort with 3+ HER2 expression received fam-trastuzumab deruxtecan-nxki, and experienced a durable partial response lasting approximately one year. Conclusions: B7-H3 and HER2 are promising ADC targets in FLC. While a subset of patients may already be eligible for fam-trastuzumab deruxtecan-nxki under its tumor-agnostic indication, B7-H3 shows particularly strong and widespread expression across both tumor and stromal compartments. These findings support further preclinical and clinical evaluation of B7-H3- and HER2-directed ADCs as targeted treatment strategies for advanced FLC. Citation Format: Waqar Arif, Elsa Hallab, Franshisca Hayek, Howard Liu Li, Mari Nakazawa, Mark E. Furth, Andrew S. Liss, Patricia Cogswell, Ezra G. Baraban, Jacqueline Birkness-Gartman, Marina Baretti, Robert A. Anders, Mark Yarchoan. Profiling the expression of antibody-drug conjugates in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2456.

Representative plots of annotated single cells delineated by IMC workflow

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense stroma and myeloid-rich microenvironment that confer resistance to immunotherapies. Previous studies demonstrated that disrupting the immune-stroma CXCR4-CXCL12 axis facilitates T cell recruitment and mobility to collaborate with anti-PD1/PD-L1 therapy. We sought to test the clinical viability of this immunotherapeutic strategy. 21 patients with metastatic PDAC were enrolled and treated in a phase 2 trial evaluating the effects of the plerixafor/AMD3100 and cemiplimab. Primary endpoint was objective response rate. Blood and tissue biospecimens were collected for correlative analyses. Parallel mouse studies were used to determine potential mechanisms of resistance. Treatments were well-tolerated, but only two patients demonstrated a best response of stable disease. Correlative analyses confirmed significant mobilization of immune cells into circulation as well as increased immune infiltration into the tumor. High-parameter imaging revealed higher levels of CD8+ T cells but also granulocytes and macrophages upon treatment. Spatial analysis showed that treatment resulted in closer proximity between macrophages and T cells but not between granulocytes and T cells. Mouse studies revealed that whereas total granulocyte depletion had no effect on immunotherapeutic efficacy, macrophage-targeting yielded significant benefit. Tumor growth measurements and immune profiling of immunotherapeutic combinations incorporating macrophage-targeting showed that despite the increased T cell infiltration, CXCR4 antagonism was in fact associated with enrichment of CD206hiIA/IElo macrophage subtypes and modestly dampened efficacy. Our findings validate the utility of CXCR4 antagonism as an effective immune-recruiting platform but also underscores the need for strategies that better leverage its effects.

Fibrolamellar carcinoma (FLC) is a rare and aggressive malignancy of the liver that predominantly affects adolescents and young adults. These tumors are distinct from conventional hepatocellular carcinomas and are characterized histologically by thick fibrous collagen bands. The transcriptome of FLC is characterized by a recurrent oncogenic fusion between DNAJB1 and PRKACA. Liver resection is a potentially curative treatment option for early-stage FLC, but there are no standard systemic therapies available for advanced FLC. Most patients present with unresectable disease or relapse after surgery; for these individuals, outcomes are dismal with a median survival of only 12 months. This highlights the urgent need to understand the natural evolution of FLC, which in turn will support the development of effective targeted therapies. To investigate the evolution of FLC, we leveraged FLC tissue samples from n=5 patients with paired primary and distant metastatic surgical biopsies. Among these patients, 4/5 received systemic therapy including immunotherapy in the interval between primary resection and resection of the distant metastatic lesions. A histological view of the cases revealed notable morphological differences between the primary and metastatic tumors. Primary tumors showed the characteristic oncocytic tumor cells separated by thick cords of collagen fibrosis. However, the metastatic lesions exhibited reduced fibrosis or lacked it entirely, suggesting malignant progression of the tumor and loss of a key histological hallmark of FLC over time. To date, most transcriptomic analysis of FLC has been performed on bulk tissue which neglects the heterogeneity of the tumor. To capture the complexity of the lesion, we performed spatial transcriptomic analysis of our paired samples (n = 10, primary and metastatic lesions) using the VisiumHD platform by 10x Genomics. Furthermore, we succeeded in labeling the neoplastic cells using a custom-designed probe against the DNAJB1-PRKACA chimeric transcript which we included in the standard probe library. Spatially resolved gene expression analysis and neighborhood analysis using the Seurat workflow revealed marked differences in transcriptional networks coinciding with the progression of the tumor. Here we present a spatially and temporally resolved transcriptome atlas of FLC. This rich and valuable resource will be critical for understanding FLC biology and ultimately developing novel therapeutics. Waqar Arif, Luciane Kagohara, Robert A. Anders, Kiyoko Oshima, Mark Yarchoan. Spatially resolved gene expression analysis of primary and metastatic fibrolamellar hepatocellular carcinoma (FLC) [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 5071.

In pancreatic ductal adenocarcinoma (PDAC), venous invasion (VI) is a critical step in metastasis and is associated with poor survival. However, little is known about the molecular features of VI. To investigate, we performed spatial transcriptomic analysis of 95 human PDAC tissue samples from eight treatment-naïve patients. Our analysis revealed that, compared with PDAC in stroma, PDAC with VI demonstrated up-regulation of genes associated with epithelial differentiation, classical subtype, and benign exocrine function. Conversely, PDAC with VI demonstrated down-regulation of genes associated with mesenchymal differentiation, basal-like subtype, and disease aggression. Additionally, we uncovered characteristics of VI morphology that correlated with these molecular features. VI-intraepithelial neoplasia-like foci had preserved venous architecture and had a classical, epithelial molecular phenotype, whereas VI-destructive foci had destroyed venous architecture and had a more basal-like, mesenchymal phenotype. We contextualized our findings using public RNA-seq data and observed that metastatic PDAC had greater similarity to PDAC in stroma than to PDAC with VI, whereas circulating tumor cells showed no preferential association. We confirmed our findings by spatial proteomic analysis of VI in an independent cohort of 19 treatment-naïve patients with PDAC. Overall, our work provides a reference atlas of spatial transcriptomics and proteomics of VI in PDAC and reveals unexpected increases in molecular features associated with better patient outcomes.

The cholangioblastic variant of intrahepatic cholangiocarcinoma is a distinctive neoplasm that typically affects young women without underlying liver disease. Morphologically, it demonstrates solid, trabecular, and tubulocystic architecture, biphasic small cell-large cell cytology, and immunoreactivity for inhibin, neuroendocrine markers, and biliary but not hepatocellular markers. In 2021, our group identified a characteristic NIPBL::NACC1 gene fusion in cholangioblastic cholangiocarcinoma, and since then ~20 genetically confirmed cases have been reported in the literature. We report 2 additional cases, both of which caused diagnostic challenges. The first was previously published as a “biliary adenofibroma with malignant features” which we now show recurred as a high-grade adenocarcinoma. Re-review of the original lesion demonstrated the morphologic and immunohistochemical features of highly cystic cholangioblastic cholangiocarcinoma, whereas the high-grade recurrence lacked many of these features. In addition to the characteristic NIPBL::NACC1 gene fusion, the recurrence demonstrated loss of the RB1 and PTEN genes which were found in the highly cystic, bland areas of the original tumor, suggesting that the recurrence was derived from this bland component. The second case was originally misclassified as metastatic well-differentiated neuroendocrine neoplasm and only focally demonstrated the characteristic biphasic small cell-large cell cytology. In addition, a review of 7 cholangioblastic cholangiocarcinomas in our files demonstrates that loss of chromosome 13q14.2 (where the RB1 gene resides) and loss of chromosome 6q15-q16.3 are recurrent secondary changes in these neoplasms. Expression profiling demonstrated alterations in the transforming growth factor receptor beta superfamily, and overexpression of MYC which was validated by immunohistochemistry. Our findings expand the morphologic and genetic spectrum of this neoplasm and provide insight into secondary genetic changes associated with progression.

Background The biological sciences are producing increasingly larger datasets for biomarker discovery. While common data models have been developed for medical terms as they relate to patient health outcomes, a data model that supports longitudinal tracking of biospecimens and relating them against an individual patient experience is a large, unmet need.Method A structure and associated taxonomy were achieved through a six-tier build in Research Electronic Data CAPture (REDCap), which organizes the complexity of the therapeutic decisions, biospecimens, and outcomes that characterize a longitudinal patient experience. Modules were developed to support export of REDCap data into a Structured Query Language (SQL) format for merging with extended biomarker data, also housed in SQL.Results The resultant AstroID resource is a relational structure for clinical and biospecimen data that meets several desired goals: searchable, flexible, generic, Health Insurance Portability and Accountability Act-compliant, auditable, and easy-to-use. The essential elements forming the core of the six-tiered build are provided, so others can readily adopt this schema, as well as an example of an extended, customized build to support biomarker discovery for patients with melanoma. Two examples where this data structure was used to support biomarker discovery and development are described, and example queries of the database are also presented. To the extent possible, the data dictionary was aligned with large data models, such as those for the National Institutes of Health’s Human Tumor Atlas Network. The structure can readily scale to accommodate thousands of patients, multimodality data, and spatial characterization of billions of cells. Radiologic imagery can also be included along with pathology imagery to support spatial studies, including artificial intelligence-driven analyses.Conclusions This effort provides a database model for investigators conducting research on large volumes of biospecimens with clinical annotation. We have now deployed this structure in our laboratories and have over 1B cells spatially mapped, each effectively tagged with the clinical information from longitudinal patient experiences. While the description uses the example of cancer biomarkers, this data structure could be used to characterize longitudinal biospecimens from any disease process. In the near future, automatic synchronization between the electronic medical record and one or more AstroID databases is anticipated.

4019 Background: Inhibitors of Programmed Cell Death Protein-1 (PD-1) have demonstrated remarkable activity in dMMR/MSI-H cancers, leading to the first tissue agnostic FDA approval for an oncologic indication. We report herein results of long-term follow up of KEYNOTE-016, the first study to demonstrate pan-tumor activity of the PD-1 inhibitor pembro in dMMR/MSI-H solid tumors. Methods: KEYNOTE-016 was a multi-center open-label phase 2 study evaluating pembro in patients with advanced colorectal cancer (CRC) (Cohort A) or non-CRC solid tumors (Cohort C) that were dMMR and had progressed after ≥1 prior line of therapy (or ≥2 prior lines for CRC). Eligible patients were age ≥ 18, and had measurable disease per RECIST 1.1. Patients with active CNS metastases, who were on immunosuppressive therapy, had autoimmune disease, or were previously treated with immune checkpoint inhibitors were excluded. Patients received pembro IV every 2 weeks until progression, intolerance, withdrawal of consent or up to a maximum of 2 years. Results: Between 9/2013 - 9/2017, 88 patients (Cohort A: 41; Cohort C: 47) enrolled at 7 sites and received ≥1 dose of pembro. Tumor types enrolled on Cohort C included endometrial (N = 15), pancreatic (N = 9), small intestinal (N = 5), gastroesophageal (N = 5), biliary (N = 4), ampullary (N = 4), and other (N = 5). Median follow up time was 49.7 mos for all patients and 99.8 mos for alive patients. Objective response rate (ORR) was 58% with 23 partial (PR) and 28 complete responses (CR). 16 patients experienced a best response of stable disease (SD) for a disease control rate of 76%. Median PFS and OS were 34.9 mos (95% CI: 14.8-NR) and 80.8 mos (95% CI: 33.2-NR) respectively. The 3-, 5-, and 10-year OS rates were 55.1%, 53.7% and 47.4% respectively. Outcomes were similar between Cohorts A and C (see Table). Conclusions: In summary, long term follow up of KEYNOTE-016 confirms high rates of durable remission from pembro in patients with dMMR/MSI-H solid tumors, with several patients remaining alive and in remission at 10+ years follow up. Responses were seen across tumor types. Clinical trial information: NCT01876511 . Results by cohort. Cohort ACRCN=41 Cohort Cnon-CRC N=47 ORR, % 56.1 59.6 PR, N (%) 11 (27) 12 (25) CR, N (%) 12* (29) 16 (34) SD, N (%) 10 (24) 6 (13) PD, N (%) 5 (12) 9 (19) NE, N (%) 3 (7) 4 (9) PFS, median months (95% CI) 38.8 (8.1-NR) 20.5 (14.3-NR) OS, median months (95% CI) 80.8 (33.2-NR) 86.4 (21.8-NR) Follow up time, median months 51.2 35.9 3-year OS rate (%) 60.5 50.3 5-year OS rate (%) 57.5 50.3 10-year OS rate (%) 47.3 47.2 *Includes 3 patients with unconfirmed CR.

2503 Background: Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of liver cancer affecting children and young adults that is driven by a chimeric protein, DNAJ-PKAc. The development of molecular inhibitors of DNAJ-PKAc has been hampered by unacceptable on-target toxicity, but the chimera results in a tumor-specific antigen (neoantigen) that may be targeted immunologically. Methods: We conducted a phase 1 clinical trial of a therapeutic vaccine targeting DNAJ-PKAc (FLC-Vac), in combination with nivolumab and ipilimumab, in children and adults with advanced FLC. The primary objectives were safety and T cell responses, defined as 2.5-fold increase of interferon gamma (IFN-γ)-producing DNAJB1-PRKACA chimera-specific T cells in the peripheral blood after week 10 (priming phase). The study was planned with 12 evaluable patients. FLC-Vac, consisting of a peptide encoding the DNAJB1-PRACA fusion plus poly-ICLC adjuvant, was administered on weeks 0, 1, 2, 3, 6, 9 during the priming phase of the study. Nivolumab, 3 mg/kg, followed by ipilimumab, 1 mg/kg, was administered every 3 weeks for 4 doses during the priming phase. After completion of the priming phase, FLC-Vac and nivolumab were continued in maintenance. Key exclusion criteria include age < 12 years and prior treatment with immune checkpoint inhibitors. The trial incorporated a safety lead-in portion in which the first 3 patients received vaccine monotherapy for 3 weeks prior to receiving combination therapy. Results: Among 16 patients enrolled, 12 completed the vaccine priming phase and were evaluable for both immunological and clinical endpoints. The median age was 24 years (range: 12-47). Grade 3 treatment-related adverse events were reported by six patients (37.5%). DNAJ-PKAc-specific T cell responses were detected in 9/12 patients after treatment. In the subset of patients who completed the initial priming phase the disease control rate (DCR) was 75% (9/12), with three partial responses (25%). All 3 responding patients are without evidence of active cancer after undergoing surgical debulking of residual disease. All patients with clinical responses also had DNAJ-PKAc-specific T cell responses, from whom we identified multiple class II-restricted T cell receptors (TCRs) with specificity for DNAJ-PKAc. Correlates of response included both functional neoantigen reactivity and changes in TCR repertoire features over time. In two patients who experienced eventual progression after initial clinical response, we found evidence that the loss of efficacy was likely due to T cell exhaustion, and in one case was restored with checkpoint rechallenge. Conclusions: Our findings demonstrate the potential for therapeutic vaccines targeting DNAJ-PKAc in FLC and suggest a rubric for evaluating effective anti-neoantigen immunity. Clinical trial information: NCT04248569 .