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ABSTRACTBackgroundIn this study, we compared outcomes of intensified myeloablative conditioning regimens using large registry data from Japan (Japanese Society for Transplantation and Cellular Therapy) and the United States (Center for International Blood and Marrow Transplant Research).MethodsAdult patients who underwent their first myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) for acute leukemia in remission between 2010 and 2018 using conditioning regimens of cyclophosphamide plus total‐body irradiation (CY/TBI), CY/TBI+cytarabine (AraC), or CY/TBI+etoposide (VP16) were included.ResultsThe acute myeloid leukemia (AML) cohort (N = 480, 38.8%) indicated that overall survival (OS) was poorer in CY/TBI+AraC (hazard ratio [HR] 1.46, p < 0.001) and CY/TBI+VP16 (HR 1.39, p = 0.059) compared to CY/TBI. Relapse was not suppressed, while treatment‐related mortality (TRM) was significantly higher (HR 1.78 and 1.74, p < 0.001 and 0.018, respectively). In the acute lymphoblastic leukemia (ALL) cohort (N = 3901, 61.2%), OS was comparable among these regimens. With intensified regimens, relapse was significantly suppressed in CY/TBI+VP16 (HR 0.74, p = 0.005), while TRM was higher (HR 1.21, p = 0.077). No interactions were observed regarding the country.ConclusionIn AML adding AraC and VP16 to CY/TBI had an adverse effect on OS. Conversely, in ALL, adding VP16 or AraC to CY/TBI did not affect survival, but the addition of VP16 reduced the risk of relapse.Clinical Trial RegistrationThe authors have confirmed clinical trial registration is not needed for this submission.

7540 Background: The combination of a proteasome inhibitor (PI) with lenalidomide (R) and dex (d) has been a common initial therapy for newly diagnosed myeloma (NDMM). We designed a randomized phase 3 trial to examine if carfilzomib (K), a next generation PI, improved progression free survival (PFS) compared with bortezomib (V) when either is combined with Rd for initial treatment of NDMM. Initial analysis at a median follow up of 15.3 months (mos) showed comparable PFS for both triplets. We present the long-term results of the trial with ~70 months median follow up. Methods: Patients (Pts) with NDMM, were randomized 1:1 to receive VRd or KRd for 36 weeks followed by a 2 nd randomization (1:1) to indefinite versus 2 yrs of R maintenance. Pts without del17p, t(14;16), t(14;20), plasma cell leukemia or high-risk GEP70 profile, were enrolled. VRd arm included V 1.3 mg/m 2 on days(d) 1, 4, 8, and 11 (d1, 8 for cycles 9-12), R 25 mg d1-14, and d 20 mg d1, 2, 4, 5, 8, 9, 11, 12 of a 3-week (wk) cycle for 12 cycles, while pts in the KRd arm received K 36 mg/m 2 d1, 2, 8, 9, 15, 16 with R 25 mg daily on d1-21 and d 40 mg wkly, in 4 wk cycles for 9 cycles. Maintenance used 15 mg R d1-21 q4 wks. Results: The study accrued 1087 pts (VRd=542, KRd=545). Median age was 65y; baseline characteristics including intent to transplant were similar across the arms. Median induction duration (mos; IQR) was 7.2 (3.4-8.9) and 8.4 (5.1-9.1) for VRd and KRd, respectively; 59.8% in VRd and 45.3% in KRd did not proceed to Step 2. Median PFS (mos) was VRd=41.9 and KRd=44.6; HR = 0.89 (0.76-1.04). Toxicity data, PFS sensitivity analyses and OS probabilities are as in the table. Conclusions: In this randomized trial, with median follow up of nearly 6 years, KRd and VRd had comparable PFS and OS in an intent to treat analysis. While similar numbers proceeded to SCT in the 2 arms, more did so during induction in the VRd arm while more patients in the KRd arm went to SCT later. VRd remains a standard triplet induction regimen in standard and intermediate risk NDMM, and a suitable backbone for 4 drug combinations. Clinical trial information: NCT01863550 . N (%) VRd(n=527) KRd(n=526) SCT anytime 186 (34.3) 183 (33.6) SCT without Step 2 registration 146 112 Median Time to SCT (mos; range) 7.7 (3.5-83.9) 10.6 (3.7-70.6) Grade 3-4 Treatment-Related Toxicity 315 (59.8) 344 (65.4) Grade 5 Treatment-Related Toxicity 2 (0.4) 9 (1.7) Grade 5 All Events 11 (2.1) 20 (3.8) Survival outcomes HR Median (95% CI) Median (95% CI) PFS Primary: PD or death within 3 months of last evaluation as events 0.89(0.76-1.04) 41.9(35.7, 50.3) 44.6(38.2, 51.9) PFS Sensitivity: All deaths as events, 0.87(0.75-1.02) 39.5(34.9, 46.0) 42.8(37.4, 49.7) PFS Sensitivity: Censor at alternate Rx 0.83(0.69-0.99) 35.0(31.3, 42.6) 38.7(34.7, 49.0) PFS Sensitivity: Event at alternate Rx 0.84(0.73-0.97) 18.0(14.2, 23.0) 24.4(20.9, 27.9) Overall survival 0.92(0.75-1.12) 119(100, NE) 118(103-NE)

e12562 Background: Previous research has suggested that breast cancer (BC) diagnosed during pregnancy or the postpartum period exhibits more aggressive clinical features, possibly due to pregnancy-related hormonal changes, immune modulation, and alterations in the mammary microenvironment compared to nulligravid or women many years out from pregnancy. Emerging data have demonstrated potential worse outcomes for patients (pts) diagnosed in post-partum. This study evaluated the influence of timing of diagnosis in relation to pregnancy history and recency of childbirth on long-term outcomes in a modern cohort of young pts with early-stage BC. Methods: Pts with stage I–III BC age < 40y in the prospective Young Women’s Breast Cancer Study (YWBCS) were categorized at diagnosis as Nulligravid (no prior pregnancy), Nulliparous (prior pregnancy without live birth), Currently Pregnant, or Parous (subdivided by time since last childbirth: within 5 years [≤ 5y PP], and 5–10 years postpartum [5–10y PP]). Baseline characteristics and outcomes were compared across BC subtypes (estrogen receptor+/HER2- [ER+/HER2-], HER2+, and triple-negative BC [TNBC]). Prognostic analyses for distant recurrence-free survival (DRFS) used Cox models. Results: 859 pts were included, 257 (30%) nulligravid, 50 (6%) nulliparous, 37 (4%) pregnant, and 515 (60%) parous (348 [68%] were ≤5y PP, and 167 [32%] were 5–10y PP). Median follow-up was 133 (6–209) months. Median age at diagnosis was lower in nulligravid pts (33 vs. 35–38 years); T1 tumors were most common in pts >5y PP (62%), and N0 status was highest in nulligravid pts (64%) (all p < 0.0001). Pregnant pts had higher rates of TNBC (27%), and T3 tumors (14%), N1 status, N1 (41%), and Grade 3 (81%) tumors compared to other groups (all p < 0.0001). Overall, only tumor size (T2-T4 vs T1, HR 2.03; 95% CI 1.37–3.03) and nodal status (N1 vs N0, HR 2.22; 1.48–3.34; N2/3 vs N0, HR 3.23; 1.96-5.33) were independently associated with worse DRFS; pregnancy history was not: nulligravid + nulliparous pts had no significant differences in DRFS compared to Pregnant (HR 1.46; 0.75–2.86), Parous <5y PP (HR 0.86; 0.58–1.26), and Parous 5–10y PP (HR 0.89; 0.54–1.48). Subgroup analyses by BC subtype (Table) and sensitivity analyses (excluding currently pregnant pts or separating nulligravid and nulliparous pts) demonstrated no significant associations between pregnancy history and DRFS. Conclusions: In this cohort study of very young patients with early breast cancer with contemporary treatment including management of breast cancer during pregnancy, pregnancy history and recency of pregnancy were not independent predictors of long-term disease outcomes. DRFS results by subgroup. Pregnancy Group N (%) HR (95% CI) ER+/HER2- Nulligravid + Nulliparous 173 (38.1) Ref Pregnant 16 (3.5) 0.93 (0.32–2.70) Parous ≤5y PP 177 (39.0) 0.77 (0.45–1.29) Parous 5–10y PP 88 (19.4) 0.78 (0.39–1.53) HER2+ Nulligravid + Nulliparous 77 (30.8) Ref Pregnant 11 (4.4) 3.27 (0.80–13.38) Parous ≤5y PP 113 (45.2) 1.50 (0.60–3.75) Parous 5–10y PP 49 (19.6) 1.45 (0.45–4.66) TNBC Nulligravid + Nulliparous 56 (36.8) Ref Pregnant 10 (6.6) 1.92 (0.61–6.09) Parous ≤5y PP 57 (37.5) 0.68 (0.28–1.63) Parous 5–10y PP 29 (19.1) 1.16 (0.43–3.14)

5064 Background: FoundationOneCDx (F1CDx) supports two FDA-approved biomarkers to guide treatment decisions for ICI for patients with mCRPC: MSI status and TMB. MSI-H and TMB-H (10+ mut/MB) have strongly overlapping prevalence. We sought to better characterize ICI outcome associations of TMB-H / non-MSI-H population and relative effectiveness of taxanes & ICI among patients who received these agents in sequence. Methods: Following a prespecified analysis plan, this study used the nationwide (US-based) de-identified Flatiron Health-Foundation Medicine mCRPC clinico-genomic database (FH-FMI CGDB), with data originating from ~280 US cancer clinics (~800 sites of care). Inclusion criteria included patients with mCRPC treated with single-agent anti-PD(L)1 therapy in the FH network between 1/1/2011 - 3/30/2024. This study used the MSI and TMB algorithms from the tissue based F1CDx. Time to next treatment (TTNT) and OS were assessed with Kaplan-Meier plots and in multivariable Cox models adjusted for ECOG performance score, socioeconomic status, prior treatment history, and baseline PSA. Among patients who received taxanes in a prior line of therapy, the effectiveness (TTNT1 vs. TTNT2) of taxane and ICI were compared. Results: Among 2995 prostate cancer tissue specimens in the database, 95 (3.1%) were MSI-H and 142 (4.7%) were TMB-H. 94 (3.1%) were MSI-H & TMB-H, 1 was MSI-H & TMB-L, and 48 (1.6%) were TMB-H & not MSI-H. Among these, 84 patients with mCRPC were treated with ICI and met inclusion criteria, including MSI-H & TMB-H (n = 30), non-MSI-H & TMB-H (n = 8), and non-MSI-H and TMB-L (n = 46). The respective median TTNT on ICI was 8.0 vs. 9.6 vs. 3 months. The respective median OS from initiation of ICI was 10.9 vs. not reached vs. 4.4 months. In multivariable models evaluating ICI only, compared to non-MSI-H & TMB-L, the MSI-H & TMB-H group had more favorable TTNT (HR: 0.20, 95%CI: 0.10 – 0.41, p < 0.001) and OS (HR: 0.33, 95%CI: 0.16 – 0.70, p = 0.004), and the non-MSI-H & TMB-H group also had more favorable TTNT (HR: 0.13, 95%CI: 0.04 – 0.45, p = 0.002) and OS (HR: 0.20, 95%CI: 0.05 – 0.75, p = 0.017). 50 of the 84 (60%) patients treated with ICI had prior mCRPC taxane treatment. Better TTNT2 on subsequent ICI vs. prior taxane was observed for MSI-H (HR: 0.49, 95%CI: 0.23 – 1.01, p = 0.051) and TMB-H (0.54, 95%CI: 0.30 – 0.98, p = 0.044), but the opposite was true for non-MSI-H and TMB-L subgroups, with significant treatment interactions for each (p = 0.0018, p = 0.00052). Conclusions: TMB-H (4.7%) is more prevalent than MSI-H (3.1%) by F1CDx in mCRPC. Non-MSI-H / TMB-H (1.6%) in the routine practice cohort have similar outcome associations on ICI to MSI-H. Both MSI-H and TMB-H by F1CDx are predictive of differential benefit for ICI vs. taxanes in later mCRPC treatment lines.

5048 Background: Ra-223, an alpha-emitting radionuclide, is the first agent of its kind approved for the treatment of mCRPC. Although a pivotal phase 3 study evaluated its short-term safety, there is a need to investigate its long-term safety. The REASSURE study prospectively examined the long-term safety of Ra-223, including secondary primary malignancies (SPMs), in a large patient (pt) population enrolled across Europe, the United States, Israel, and Latin America. Methods: We report final analyses (data cut-off Oct 24, 2024) of REASSURE (NCT02141438), a global, noninterventional study (enrolment 2014–2017). Primary outcomes were the incidence of SPMs, short- (30 days) and long-term (7 years) safety events, and bone marrow suppression (BMS) management in pts who had ≥1 Ra-223 dose. Secondary outcomes included overall survival (OS). Results: Analyses included 1472 pts; median follow-up was 17 months (range 0.3–95.4). Median age was 73 years and 80% of pts had an ECOG PS of 0/1. In evaluable pts, median alkaline phosphatase, prostate-specific antigen, and lactate dehydrogenase levels were 133 U/L, 59 ng/mL, and 266 U/L, respectively. Overall, 81% of pts had bone-only metastases at baseline; 19% of pts had metastases in the bone plus other sites (mostly lymph nodes). Prior treatments included abiraterone (48% of pts), enzalutamide (39%), docetaxel (39%), and cabazitaxel (9%). Pts received a median of 6 Ra-223 doses; 67% received ≥5 doses. SPMs occurred in 2% of pts (25 SPMs in 24 pts). Of these pts, 16 (67%) and 1 (4%) had received prior or concomitant radiotherapy, respectively. Overall, 3% of pts had drug-related serious adverse events >30 days after completing Ra-223. Fractures were reported in 10% of pts and were less common in pts with (7% of 605) than without (12% of 867) concomitant BHA use. During Ra-223 and up to 30 days after the last dose, there was no notable difference in the incidence of abnormal platelet counts between pts with (3%) or without (2%) prior chemotherapy; similar findings were seen for abnormal neutrophil counts (5% and 5%, respectively). BMS treatments, assessed from the start of Ra-223, were more common in pts who had received prior taxanes (38%) than in those who had not (26%). The most common life-prolonging therapies received after Ra-223 were docetaxel (18%), enzalutamide (15%), abiraterone (11%), and cabazitaxel (11%). Median OS was 15.6 months (95% CI, 14.6, 16.4); pt subgroups that survived the longest will be characterized and presented. Conclusions: This real-world safety analysis of pts with mCRPC is the longest follow-up of a radiopharmaceutical reported to date and supports the well-established favorable safety profile of Ra-223. The incidence of SPMs was low. The rate of fracture was low, especially in the presence of BHAs. Prior taxane chemotherapy use had no impact on hematological toxicity. Clinical trial information: NCT02141438 .

3040 Background: EVEREST-2 is a first-in-human, phase 1/2 trial to assess the safety and efficacy of A2B694, an autologous, logic-gated Tmod CAR T therapy targeted to MSLN, which is normally expressed in the mesothelium and can be upregulated in many solid tumors. A2B694 is designed to overcome challenges of on-target, off-tumor toxicity that have limited other MSLN-targeted approaches by combining a CAR-activating receptor targeting MSLN with a blocker CAR that recognizes HLA-A*02, to distinguish between normal and tumor cells (Tokatlian, et al. J Immunother Cancer. 2022). Methods: Adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression who have progressed after standard-of-care therapy are eligible for EVEREST-2. Enrollment to EVEREST-2 and collection of T-cells occurs through the ongoing prescreening study BASECAMP-1 (NCT04981119). When clinically appropriate, A2B694 is manufactured from cryopreserved T cells, and patients undergo lymphodepletion before A2B694 infusion. The dose-escalation phase is evaluating the safety and tolerability of A2B694 to identify the recommended phase 2 dose (RP2D). Dose escalation was started at 1x10 8 Tmod positive cells (dose level [DL] 1) and will increase up to 14x10 8 in combination with low-dose IL-2 (DL 5). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Results: As of January 15, 2025, 5 participants (median age: 60 years; range, 50-84) have enrolled on EVEREST-2 and received A2B694 at DLs 1-2; participants had ovarian cancer (n = 3), pancreatic cancer (n = 1), and non-small cell lung cancer (n = 1) and had received a median of 4 prior lines of therapy (range, 1-7). Lymphodepleting chemotherapy was well tolerated with no significant cytopenias observed. The most common adverse events were lymphopenia (7 [14.6%]) and decreased appetite (6 [12.5%]). There were no dose-limiting toxicities, cytokine release syndrome, nor related neurotoxicity. One participant was admitted to the hospital for decreased appetite. No long-term toxicities have been noted up to 7.5 months post-infusion. Of the participants who have received A2B694, 5 were efficacy evaluable at DLs 1-2. A2B694 was detected post-infusion in the peripheral blood in all patients. Additionally, A2B694 was detected in an abdominal tumor biopsy from a patient with pancreatic cancer 42 days post-infusion. Conclusions: The logic-gated approach was successful at reducing toxicity seen with prior MSLN-targeted CAR T therapies, and A2B694 showed successful CAR T expansion and tumor infiltration. The maximum tolerated dose has not been reached, and results from the dose-escalation phase continue to determine the RP2D. Clinical trial information: NCT06051695 .

4020 Background: In previously treated patients (pts) with advanced extra-pancreatic NET (epNET) or pancreatic NET (pNET), cabozantinib (cabo) demonstrated improved progression-free survival compared to placebo (pbo) in the CABINET trial (NCT03375320). Here we present HRQOL data from CABINET. Methods: Pts with previously treated unresectable, locally advanced or metastatic epNET or pNET were randomized (2:1) in separate cohorts to receive cabo 60 mg daily vs pbo. HRQOL was an exploratory endpoint measured using the EORTC QLQ-C30, QLQ-GI.NET21, Patient Global Impression of Change (PGIC), and PRO-CTCAE questionnaires. Optional participation was by paper surveys at baseline and every 12 weeks (wks) until disease progression or new anticancer therapy. Mean changes from baseline were compared between treatment arms at wk12 using general linear mixed models. Minimally important within-arm improvements and between-arm differences were defined as 8 points on the C30 Global Health Status (GHS)/QOL Scale. PGIC was dichotomized as improved vs unchanged/worsened and compared using Chi-squared tests. Rates of pt-reported adverse events (AEs) by PRO-CTCAE were baseline-adjusted and compared using Fisher’s exact tests. Results: 172 pts (113 cabo, 59 pbo) with epNET and 81 pts (53 cabo, 28 pbo) with pNET consented for survey participation. Pts completed 524 (82%) of 640 expected surveys across baseline, wk12, and wk24. For epNET, C30 GHS/QOL was significantly improved at wk12 in pts receiving cabo (mean change 9.5, standard error (SE) 2.2, p < 0.001) but not pbo (mean change 0.2, SE 3.1, p = 0.95) resulting in significantly different mean changes from baseline between arms (p = 0.02). For pNET, C30 QHS/QOL was significantly improved at wk12 in pts receiving cabo (mean change 8.0, SE 3.0, p = 0.008) but not pbo (mean change 6.8, SE 4.4, p = 0.12); mean changes from baseline did not statistically differ between arms (p = 0.82). No significant differences in mean changes from baseline at wk12 in C30 Physical, Role, Emotional, Cognitive and Social Function were observed between arms in the epNET or pNET cohorts. In both cohorts at wk12, pts on cabo reported improved overall status as measured by PGIC more often than those on pbo (epNET: 38% vs 19%, p = 0.04; pNET: 57% vs 18%, p = 0.006). Rates of pt-reported AEs were statistically significantly higher in cabo for diarrhea (68% vs 53%), hand-foot syndrome (57% vs 26%), mouth or throat sores (47% vs 27%), and dysgeusia (67% vs 37%) [all p < 0.05]. GI.NET21 data will be presented. Conclusions: Although cabo is associated with pt-reported AEs consistent with its known safety profile, global HRQOL was maintained. A higher proportion of patients receiving cabo reported improved overall status. Results support cabo as a treatment option for pts with previously treated advanced NET that preserves patient HRQOL. Clinical trial information: NCT03375320 .

9503 Background: Neoadjuvant ± adjuvant immunotherapy improves event-free survival relative to adjuvant immunotherapy alone for patients with high-risk resectable stage III melanoma. However, the optimal regimen balancing efficacy and tolerability is not known. T-cell immunoglobulin and ITIM domain (TIGIT) is a promising immune checkpoint but its therapeutic potential in stage III melanoma is underexplored. Methods: In this phase II trial, patients with resectable, macroscopic stage III melanoma received four 21-day neoadjuvant cycles of 1200mg IV atezolizumab (atezo, anti-PD-L1) + 600mg IV tiragolumab (tira, anti-TIGIT), followed by therapeutic lymph node dissection (TLND) and eight 21-day adjuvant cycles of 1200mg IV atezo. Primary endpoints were pathologic response (of all patients initiating neoadjuvant therapy) and recurrence-free survival (RFS) from the time of TLND in patients who were operated on per protocol and received adjuvant therapy. Secondary endpoint was adverse events (AEs); exploratory endpoints included event-free survival (EFS) and distant metastasis-free survival (DMFS). Results: Thirty-four patients, median age 59 years, were accrued and initiated neoadjuvant atezo/tira. 76.5% had >1 metastatic lymph node involved at baseline and 73.5% presented with Stage IIIC disease. All 34 patients were evaluable for AEs, pathologic response and EFS. Four patients were diagnosed with metastatic disease during neoadjuvant treatment, while 30 had TLND per protocol and 28 received adjuvant treatment and were evaluable for RFS and DMFS. Major pathologic responses (MPR, ≤10% viable tumor) were observed in 16/34 patients (47.1%, Table). With 19.9 months median follow-up from registration, 12-month EFS was 72.0% (95% CI 57.9 to 89.5%). With 16 months median follow-up from operation, 12-month RFS was 73.3% (n=28, 95% CI 56.9 to 94.5%), while 12-month DMFS was 86.0% (n=28, 95% CI 72.2 to 100%). In the 16 patients with an MPR, 2 did not receive adjuvant treatment and were followed for 33.3 and 10.8 months without recurrence/death. In the remaining 14 patients, 12-month RFS and 12-month DMFS were both 91.7% (95% CI 77.3 to 100%). 5 patients (14.7%) experienced any grade 3+ AE with 2 (5.9%) at least possibly related to the neoadjuvant regimen. Conclusions: Among patients with high-risk resectable stage III melanoma, neoadjuvant atezo/tira was a promising regimen with a favorable safety profile and warrants further study. Identification of predictive biomarkers will allow for optimization of neoadjuvant therapy for individual patients. Clinical trial information: NCT03554083 . Pathologic response. N=34 Major Pathologic Response 16 (47.1%) Pathologic complete response (no viable tumor) 13 (38.2%) Near-pathologic complete response (0.1−10% viable tumor) 3 (8.8%) Pathologic partial response (>10.0−50% viable tumor) 2 (5.9%) Pathologic non-response (>50% viable tumor) 12 (35.3%) No per protocol operation 4 (11.8%)

2030 Background: Brain metastases (BM) are common and arise in 30% of lung adenocarcinoma (LUAD) patients. Patients with LUAD that develop BM have significantly poorer outcomes, with a 10-16 month median overall survival. Unfortunately, current clinical practice for BM prediction is limited and so BM are typically detected after they develop and grow to cause neurological symptoms. Once BM are detected, currently neurosurgical tumor biopsies are performed to enable BM diagnosis via neuropathological evaluation. The aims of this study were to develop DNA methylation-based models that predict LUAD BM and non-invasively detect BM in blood to enable early diagnosis and treatment. Methods: DNA methylomes were acquired from 402 tumor tissue and plasma samples in a cohort of 346 LUAD and BM patients. Machine learning models were built using DNA methylation signatures that stratify BM risk in tissue and detect BM in plasma. Models were evaluated in independent validation datasets. A predictive nomogram was developed using the BM prediction model together with clinical factors to provide composite patient-specific scores reflecting BM risk. Results: The methylation-based BM predictor accurately stratified BM risk in a univariable Cox model using validation set data (HR = 5.65, 95%CI 1.85–17.2, p = 0.0023). Model utility was independent of the predictive value of clinical factors in a multivariable Cox model using validation set data (Table 1: HR = 8.92, 95%CI 1.97–40.5, p = 0.0046). The 5-year model accuracy was 0.81 and significantly higher than a similarly built cancer stage-based model (0.65), demonstrating utility over current practice. The combinatorial clinical-methylomic predictive nomogram had enhanced utility with an accuracy of 0.82 univariable Cox HR of 17.2 (95%CI 4.13–71.3, p < 0.0001), demonstrating comprehensive patient-specificity. The plasma-based model accurately classified BM from gliomas and lymphomas (AUROC=0.80), as typical clinical differential diagnoses, in validation set data. The models were validated further in additional external data. Conclusions: DNA methylation-based modeling of BM can accurately predict LUAD patients at risk for BM development and can non-invasively detect BM that develop. Future treatment approaches may tailor initial LUAD treatment and ongoing cancer surveillance to a patient’s BM risk, allowing for the potential to prevent and treat BM early. DNA methylation-based BM prediction is independent of clinical factors in a multivariable Cox proportional hazards model. Variable HR 95% CI p Methylome risk score 8.92 1.97–40.5 0.005 Age Years 0.96 0.92–1.02 0.177 Smoking Pack-years 0.99 0.95–1.03 0.496 EGFR Mutant vs wildtype 0.92 0.25–3.34 0.895 T T2 vs T1 1.58 0.41–6.04 0.505 T3−4 vs T1 1.49 0.28–7.98 0.642 N N1 vs N0 1.05 0.31–3.58 0.943 N2−3 vs N0 1.00 0.27–3.69 0.995 M M1 vs M0 145 12.2–1730 <0.001

TPS2094 Background: Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating tumor (ct) and cell-free (cf) DNA levels in blood due to the blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with the development and use of biomarker-driven systemic therapies. Low intensity focused ultrasound combined with intravenously administered microbubble oscillators (MB-FUS), leads to non-invasive BBB opening. This trial aims to evaluate the utility of LIFU for bolstering blood ctDNA and cfDNA for enhance liquid biopsy in patients with GBM. Methods: LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial evaluating safety and technical efficacy of transcranial MR-guided MB-FUS for increasing blood ctDNA and cfDNA levels in adults, aged 18-80 years with GBM. Patients with suspected GBM planned for tumor biopsy or resection at 17 centers in US and Canada are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication, after which MR-guided BBB opening using a 220 kHz device, with 1024-element phased array transducer, is performed with real-time acoustic feedback control for effective cavitation. Pre- and post-procedure, phlebotomy and MRI brain are done. Patients are offered optional 2 nd procedure during adjuvant chemotherapy phase if willing. Primary efficacy endpoint is correlation between biomarker patterns in tumor tissue collected during surgery/biopsy and blood collected following MB-FUS procedure. Confirmatory secondary efficacy endpoint is ratio between greatest yield of cfDNA in blood post-MB-FUS compared to cfDNA level in blood pre-MB-FUS. The primary study hypothesis is that agreement rate on biomarker pattern between resected/biopsied tumor tissue and blood is > 70%. The secondary hypothesis is that MB-FUS BBBO leads to a ≥2-fold rise in blood cfDNA. Assuming the true agreement rate expected is 89%, a sample of N = 50 patients will provide 90% power to meet the primary endpoint (Exact test, Binomial Proportion, one-sided Alpha = 0.025). Exploratory endpoints include (1) sensitivity of detection of known specific somatic mutations in ctDNA from blood samples collected before and after MB-FUS, (2) estimation of ctDNA levels in samples collected at 30-minutes, 1-hour, 2-hour, and 3-hour post-MB-FUS to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBB opening and ctDNA-based biomarkers from post-MB-FUS blood samples, (4) biomarker correlation between plasma cfDNA sampled during adjuvant chemotherapy phase and tumor tissue harvested at surgery. Patient enrollment commenced in 2022 and is ongoing (NCT05383872). Clinical trial information: NCT05383872 .