Chen, Ruqin

Summary Functional ex vivo assays using live tumor tissues have demonstrated strong predictive accuracy for response to immune checkpoint inhibitors (ICIs) but are not scalable, requiring manual processing of large resections collected at academic centers. Here, an ex vivo live tumor fragment (LTF) platform was developed using standard-of-care biopsies from 228 patients with suspected malignancy collected across prospective, multicenter observational trials and biobanks. Hierarchical clustering of ICI-mediated changes in cytokine production identified two groups: responders and nonresponders. A binary classifier (elive index) using 8 cytokines achieved an AUC of 0.99 for cluster prediction. elive index correctly predicted clinical benefit in 93% (26/28) of patients ( P = 3.2x10 -5 ) and accurately identified 83% (10/12) of objective responders. Critically, elive responders were identified among biomarker-negative patients, highlighting the platform as a scalable approach that complements existing companion diagnostics and expands the population of patients identified to benefit from ICI therapy.

9558 Background: Ipilimumab 3 mg/kg plus nivolumab 1 mg/kg (Ipi3+Nivo1) is the standard immunotherapy regimen for metastatic melanoma with brain metastases (MBM) but is associated with significant immune-related toxicity, particularly neurologic toxicity. Ipilimumab 1 mg/kg plus nivolumab 3 mg/kg (Ipi1+Nivo3) has demonstrated improved tolerability in extracranial melanoma, though comparative data in MBM are limited. We compared efficacy and toxicity of these dosing strategies in a multisite real-world cohort. Methods: We retrospectively identified patients with MBM treated with ipilimumab plus nivolumab across three Mayo Clinic sites between 2016 and 2024. Patients were classified by induction dosing as Ipi1+Nivo3 or Ipi3+Nivo1. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan–Meier method and compared using log-rank tests. Best overall response (BOR) and toxicity outcomes were summarized descriptively. Results: Among 147 patients (Ipi1+Nivo3 n=32; Ipi3+Nivo1 n=115), 144 were evaluable for survival. Median age was higher in the Ipi1+Nivo3 group (69.5 vs 66.0 years); males predominated in both groups (71.9% vs 61.7%), and the cohort was predominantly White (>96%). Most patients had ECOG 0–1 in both groups (84.4% in Ipi1+ Nivo3 and 90.4% in Ipi3+Nivo1). BRAF mutation status was balanced between regimens, with approximately half of patients harboring BRAF-mutant disease (51.6% vs 53.2%). Median OS was 18.5 months (95% CI, 5.7–54.7) with Ipi1+Nivo3 versus 10.6 months (95% CI, 6.5–19.1) with Ipi3+Nivo1 (log-rank p=0.25). Two-year OS was 45.9% and 35.0%, and five-year OS was 24.5% and 19.2%, respectively. Median PFS was 7.3 months (95% CI, 1.8–10.9) versus 2.8 months (95% CI, 2.1–4.4) (log-rank p=0.16). BOR was available for 138 patients; objective response rates were 35.5% and 31.8%, respectively. 3–5 adverse events occurred in 24.1% of Ipi1+Nivo3 versus 30.5% of Ipi3+Nivo1 patients. Hospitalizations were less frequent (16.7% vs 36.1%), and neurologic adverse events occurred only with Ipi3+Nivo1 (11.9% vs 0%). Conclusions: In this multisite real-world MBM cohort, Ipi1+Nivo3 demonstrated comparable efficacy, numerically longer survival, and a more favorable toxicity profile compared with standard Ipi3+Nivo1. Notably, no neurologic toxicity was observed with the Ipi1+Nivo3 regimen. These findings support further prospective evaluation of lower dose ipilimumab strategies. Response and survival outcomes. Outcome Ipi1 + Nivo3 Ipi3 + Nivo1 Complete response (CR) 2 / 31 (6.5%) 8 / 107 (7.5%) Partial response (PR) 9 / 31 (29.0%) 26 / 107 (24.3%) Objective response rate (ORR = CR+PR) 11 / 31 (35.5%) 34 / 107 (31.8%) Disease control rate (DCR = CR+PR+SD) 18 / 31 (58.1%) 51 / 107 (47.7%) Median PFS, months (95% CI) 7.3 (1.8–10.9) 2.8 (2.1–4.4) 2-year PFS 27.6% 22.3% Median OS, months (95% CI) 18.5 (5.7–54.7) 10.6 (6.5–19.1) 2-year OS 45.9% 35.0% 5-year OS 24.5% 19.2%

9551 Background: Lymphodepleting (LD) chemotherapy is required prior to tumor-infiltrating lymphocyte (TIL) therapy, yet the depth, kinetics, and completeness of peripheral T-cell recovery in practice remain poorly defined. Using a pooled, multi-institution cohort, we characterized longitudinal CD4 and CD8 T-cell reconstitution and the prevalence of CD4 lymphopenia following TIL therapy. Methods: We conducted a retrospective multicenter study across Mayo Clinic, Moffitt Cancer Center, and Massachusetts General Hospital of patients with metastatic melanoma treated with TIL (N = 41, median follow-up 7.6 months). Absolute CD4 and CD8 T-cell counts and CD4:CD8 ratios were assessed at baseline (pre-LD), week 4, month 3, and month 6 post-TIL. Clinically significant CD4 lymphopenia was defined as CD4 < 200 cells/µL. All patients received Pneumocystis jirovecii and antiviral prophylaxis, with antibacterial and antifungal prophylaxis administered per institutional protocols. Post-TIL infections were recorded. Paired changes from baseline were evaluated using Wilcoxon signed-rank testing. Results: Median age was 65 years (range 35-79), and 68% were male. At baseline, 16 patients (39%) had CD4 counts below the institutional lower limit of normal prior to LD. Eighteen patients (44%) received bridging therapy prior to TIL and had lower baseline CD4 and CD8 counts compared with those who did not ( p < 0.05 for both); however, post-TIL CD4 and CD8 recovery trajectories did not differ between groups. CD4 counts declined sharply following LD and demonstrated incomplete and heterogeneous recovery. Median CD4 decreased from 490 cells/µL at baseline to 250 at week 4, 290 at month 3, and 220 at month 6, remaining significantly below baseline at all post-TIL timepoints ( p < 0.05). Clinically significant CD4 lymphopenia was observed in 45% (10/22) of evaluable patients at month 3 and in 50% (44%, 7/16) at month 6. In contrast, CD8 counts increased early after TIL (median 240 to 590 cells/µL at week 4; p = 0.004) and remained higher than baseline at month 6. The CD4:CD8 ratio inverted from a median of 2.02 at baseline to 0.46 at week 4 and remained below 1.0 through month 6, reflecting sustained CD4 suppression despite CD8 recovery. Post-TIL infections occurred despite widespread prophylaxis, including bacterial (13%), viral (10%), and fungal (4.9%) infections. Conclusions: In a multi-institution cohort, LD chemotherapy preceding TIL is associated with profound and prolonged CD4 T-cell suppression, persistent CD4:CD8 inversion, and a high prevalence of CD4 < 200 cells/µL through 6 months. These findings identify delayed immune reconstitution as a common and clinically relevant consequence of TIL therapy and support prospective optimization of antimicrobial prophylaxis duration, vaccination timing, and immune monitoring strategies.

2646 Background: Immune checkpoint inhibitors (ICIs) have transformed the management of many advanced cancers but identifying patients who are likely or unlikely to benefit from ICI treatment remains a critical challenge. Biomarkers, such as PD-L1, mismatch repair deficiency, and tumor mutational burden, capture only static, unidimensional features of the complex tumor microenvironment, and therefore inadequately predict response to immunotherapy in patients. In contrast, live tumor fragments (LTFs) preserve the full complexity of the tumor microenvironment and enable functional ex vivo assessments of ICI activity. Previous efforts have had limited clinical applicability because they required larger resection specimens often collected at a single academic center. Here, the predictive capability of an ex vivo platform using core needle and forceps biopsies from multiple clinical sites was assessed and compared to approved biomarkers. Methods: Patients with eligible solid tumors (where ICI has an indication, including renal, non-small cell lung, bladder, colorectal, and triple-negative breast cancer, and others) were enrolled in ongoing, prospective observational trials (NCT05478538, NCT05520099, NCT06349642). Biopsies were cut into LTFs, encapsulated in hydrogel, and treated sequentially with IgG antibody followed by ICI (αPD-[L]1 with or without αCTLA-4). Cytokine production was assessed by a multiplexed bead-based immunoassay at multiple time points during ex vivo treatment. Using receiver operating characteristic and precision recall analyses, we identified 9 predictive cytokines (including IFN-γ, granzyme B, and CXCL9/10), which were used to develop the Elephas score (ELP-score) to assess cytokine response to ex vivo ICI treatment. Results: Of 167 eligible patient specimens, 85% (n = 142) passed quality control metrics and 130 were used for model development. Hierarchical clustering of cytokine production revealed that 27% (n = 35) of patient specimens exhibited a cytokine response to ICI. In a validation set of 20 tumors, collected from patients who were subsequently treated with ICI, ELP-score positivity correctly identified 9 of 11 patients (82%) who had an objective response (PR/CR) to ICI, including 2 patients who were negative for FDA-approved standard-of-care ICI biomarkers. Furthermore, 5 of 5 patients (100%) with clinical progressive disease were correctly characterized as ELP-score negative. Conclusions: Using routine biopsy specimens, the ex vivo live tumor platform accurately identifies patients with clinical response to ICI therapy, including those incorrectly identified by conventional biomarkers. Ongoing efforts will test these findings in an additional validation set. If confirmed, the ex vivo LTF platform could enable the identification of patients likely to benefit from ICI therapy. Clinical trial information: NCT05478538 , NCT05520099 , NCT06349642 .