Baseline Biopsies Research Articles

Safety, Efficacy and Biomarker Analysis of Crizotinib in MET Mutated Non-Small Cell Lung Cancer - Results from the Drug Rediscovery Protocol.

MET mutations occur in 3-4% of advanced non-small cell lung cancer (aNSCLC), correlating with poor survival. Despite known sensitivity of MET mutated (METmut) aNSCLC to c-MET-inhibition, no approved therapies existed until 2022. In the Drug Rediscovery Protocol (NCT0295234), patients with an actionable molecular profile are treated with off-label registered drugs. Both treated and untreated patients with aNSCLC harboring MET exon 14 skipping (METex14) or other METmuts received crizotinib 250 mg BID until disease progression or intolerable toxicity. Primary endpoints were clinical benefit (CB: RECIST v1.1 confirmed partial response (PR), complete response (CR) or stable disease (SD) ≥16 weeks) and safety. Patients were enrolled using a Simon-like two-stage design, with eight patients in stage 1 and if ≥1/8 patients had CB, 24 patients in stage 2. Whole genome and RNA-sequencing were performed on baseline biopsies. Between 09/2018 and 10/2022, 30 patients started treatment, and 24 were response-evaluable after completing ≥1 full treatment cycle. Two patients (8.3%) achieved CR, thirteen (54.2%) PR and two (8.3%) SD. The CB-rate was 70.8% (95%CI 48.9-87.4) and the objective response rate was 62.5% (95%CI 40.6-81.2). After 21.2 months median follow-up, median duration of response, progression-free and overall survival were 9.3 (95%CI 6.5-NA), 10.2 (95%CI 6.0-20.1) and 13.0 months (95%CI 9.0-NA), respectively. Twenty-three treatment-related grade ≥3 adverse events occurred in 12/30 patients (40%), causing treatment-discontinuation in three (10%). One patient (achieving CR) had a tyrosine kinase domain mutation (p.H1094Y), all other patients had METex14. Crizotinib is a valuable treatment option in METmut aNSCLC.

Does old-to-young kidney transplantation rejuvenate old donor kidneys?

The number of older organ donors is increasing due to the aging population. Aged kidneys often face problems such as delayed graft function but previous murine experiments suggested the possibilities of rejuvenation, for example, in a parabiosis setting between old and young mice. To investigate kidney-graft rejuvenation, we compared an old-to-young (O-Y) patient transplantation group and a transplantation group with donors/recipients of approx. the same age (SA) with the renal senescence marker p16 in kidney biopsy samples at baseline and one year post-transplantation. We retrospectively analyzed our hospital's 32 cases of living-donor ABO-compatible transplants performed between 2013-2020. Both the baseline and one-year biopsy (n=9) or only the baseline biopsy (n=32) were analyzed. We divided the nine cases into an O-Y group (donors' median age 68 yrs, recipients 41, difference -27) and an SA group (donors' median age 53 yrs, recipients 51.5, difference -3.5). p16 was stained with the clones JC8 and E6H4 to determine the precise p16-positive rate. The 32 baseline biopsies' p16-positive rate was weakly related to donor age, suggesting that the p16-positive rate can help evaluate kidney senescence. The (n=5) O-Y group's p16-positive rates were at baseline 0.08 and one year 0.12; the (n=4) SA group's rate was 0.03 at both baseline and one year. No kidney rejuvenation was observed, even when old donor kidneys went to young recipients.

Comparison of clinicopathological characteristics, efficacy of neoadjuvant therapy, and prognosis in HER2-low and HER2-ultralow breast cancer

BackgroundThis study aims to analyze potential differences in clinicopathology, efficacy of neoadjuvant therapy (NAT), and clinical outcome among HER2-null, HER2-ultralow and HER2-low breast cancers.MethodsConsecutive cases of HER2-negative breast cancer that received NAT were included. They were classified as HER2-null (no staining), HER2-ultralow (incomplete faint staining in ≤ 10% of tumour cells) and HER2-low (HER2-1 + or HER2-2+, in situ hybridisation negative). Subgroup analysis was performed based on the HER2 expression level.ResultsOut of 302 patients, 215 (71.19%) were HER2-low, 59 (19.54%) were HER2-ultralow, and 28 (9.27%) were HER2-null. In comparison to the HER2-ultralow group, the HER2-low group exhibited higher expression frequencies of ER (p < 0.001), PR (p < 0.001), and AR (p = 0.004), along with a greater prevalence of the luminal subtype (p < 0.001). The HER2-ultralow group also demonstrated a higher prevalence of lymph node metastasis compared to the HER2-null group (p = 0.026). Varied rates of pathologic complete response (pCR) were observed among the three subgroups: HER2-null, HER2-ultralow, and HER2-low, with rates of 35.71%, 22.03%, and 12.56%, respectively. Only the HER2-low subgroup exhibited a significant difference compared to HER2-null (p = 0.001). Despite variations in pCR rates, the three subgroups exhibited comparable disease-free survival (DFS) (p = 0.571). Importantly, we found HER2-low patients with better treatment response (RCB-0/I) exhibited significantly better DFS than those with significant residual disease (RCB-II/III) (P = 0.036). The overall rate of HER2 immunohistochemical score discordance was 45.24%, mostly driven by the conversion between HER2-0 and HER2-low phenotype. Notably, 32.19% of cases initially classified as HER2-0 phenotype on baseline biopsy were later reclassified as HER2-low after neoadjuvant therapy, and it is noteworthy that 22 out of these cases (78.57%) originally had an HER2-ultralow status in the pretreatment biopsy sample.ConclusionsOur results demonstrate the distinct clinicopathological features of HER2-low and HER2-ultralow breast tumors and confirm that RCB is an effective predictor of prognosis in HER2-low populations for the first time. Notably, our findings demonstrate high instability in both HER2-low and HER2-ultralow expression from the primary baseline biopsy to residual disease after NAT. Furthermore, this study is the first to investigate the clinicopathological feature and the effectiveness of NAT for HER2-ultralow breast cancer.

PI3K/PTEN/mTOR pathway dynamic tracking and prognostic value in HR+/HER2− BC patients with residual disease after neoadjuvant chemotherapy: a cohort study

AimsHormone receptor-positive (HR)+/HER2− breast cancer (BC) is highly heterogeneous, with PI3K/PTEN/mTOR pathway alterations emerging as possible players within this complexity. We longitudinally tracked PI3K/PTEN/mTOR pathway dynamics from baseline biopsy to...

High Mechanical Conditioning by Tumor Extracellular Matrix Stiffness Is a Predictive Biomarker for Antifibrotic Therapy in HER2-Negative Breast Cancer.

Tumor progression has been linked to stiffening of the extracellular matrix caused by fibrosis. Cancer cells can be mechanically conditioned by stiff extracellular matrix, exhibiting a 1,004-gene signature [mechanical conditioning (MeCo) score]. Nintedanib has demonstrated antifibrotic activity in idiopathic pulmonary fibrosis. This study explores nintedanib's antifibrotic effect on breast cancer outcomes. We present long-term follow-up and analysis of a neoadjuvant randomized phase II trial in early HER2-negative breast cancer. Patients (N = 130) underwent a baseline biopsy and received 12 paclitaxel courses alone (control arm) or in combination with nintedanib (experimental arm). The tumor MeCo score was determined by RNA sequencing. The primary aim was to assess nintedanib's impact on event-free survival based on MeCo scores. Follow-up data were retrieved from 111 patients; 75 baseline and 24 post-run-in phase samples were sequenced. After median follow-up of 9.67 years, median event-free survival was not statistically different between arms (P = 0.37). However, in the control arm, high- versus low-MeCo patients had a statistically higher relapse risk: HR = 0.21; P = 0.0075. This risk was corrected by nintedanib in the experimental arm: HR = 0.37; P = 0.16. Nintedanib demonstrated pharmacodynamic engagement, reducing the MeCo score by 25% during the run-in phase (P < 0.01). Patients with low MeCo after run-in had the best long-term prognosis (HR = 0.087; P = 0.03). High MeCo is predictive of poor outcomes in HER2-negative early breast cancer, although this risk can be mitigated by nintedanib, which is able to specifically reduce MeCo.

Abstract A004: A Phase 1B/2 trial of second line immunotherapy with pepinemab and avelumab for patients with metastatic pancreatic adenocarcinoma

Abstract Background: Pancreatic adenocarcinoma (PDAC) is a leading cause of cancer-related mortality with poor prognosis despite maximal therapy and is recalcitrant to approved immunotherapies. In PDAC, Semaphorin 4D (SEMA4D) expression is prognostic in patients who have undergone surgical resection and correlates with tumor infiltration of activated CD8+ T-cells. Furthermore, preclinical studies suggest that monoclonal blockade of SEMA4D supports CD8+T cells tumoral infiltration and activation, sensitizing PDAC to immune checkpoint blockade (ICB). Thus, we hypothesized that a humanized anti-SEMA4D antibody (pepinemab) may improve disease control and responsiveness to ICB in patients with PDAC. Methods: We designed a phase Ib/II single-arm clinical trial to evaluate the safety, tolerability, and efficacy of pepinemab with anti-PD-L1 (avelumab) in patients with chemotherapy-refractory PDAC (NCT05102721). The study follows a dose de-escalation schema starting at a dose combination of 20mg/kg pepinemab and 800mg avelumab every two weeks. Patient accrual for phase 1b utilizes the Bayesian Optimal Interval design targeting a dose-limiting toxicity rate of 30% or less. After 16 subjects receive a given combination dose, a Simon’s two stage assessment of futility will be undertaken with expansion to phase 2 if 2 or more subjects demonstrate response. Treatment response is assessed via RECIST 1.1 criteria with surveillance CT prior to enrollment and after completion of two cycles (8 weeks). Baseline and on-treatment tumor biopsies (after completion of one cycle, 4 weeks) are performed to analyze changes in immune, stromal, and genomic profiles to elucidate mechanisms of treatment response and failure. Patient reported outcomes are incorporated (FACT-Hep and FAACT subdomains) to assess disease-specific symptoms. Results: A total of 14 patients have been enrolled to date. Three subjects withdrew from the study prior to completing the evaluable period (four weeks of treatment) and nine subjects terminated treatment due to disease progression. Two subjects continue to receive combination treatment pending evaluation of tumor response. Baseline and on-treatment biopsies have been obtained and stored for correlative analysis from 8 evaluable subjects. One treatment-related adverse event occurred though the patient was able to complete treatment without further events. No treatment-related toxicities have been encountered. Conclusions: Combining pepinemab with avelumab appears to be safe and tolerable to date. Nine of eleven patients demonstrated disease progression, though 1 of these 9 demonstrated mixed response with some lesions showing radiologic partial and even complete response. Overall treatment response, survival data, and correlative science forthcoming. Citation Format: Luis I Ruffolo, Matthew Byrne, Bailey Hilty, Brian A Belt, Yatee Dave, Paul Burchard, Terrence Fisher, Elizabeth E. Evans, Crystal Mallow, Megan Boise, Darren Carpizo, Jen Jen Yeh, David C Linehan, Daniel Mulkerin. A Phase 1B/2 trial of second line immunotherapy with pepinemab and avelumab for patients with metastatic pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A004.

Abstract C073: FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort

Abstract Introduction: Epithelial-mesenchymal transition (EMT) is one of the key drivers of aggressive biology and development of chemoresistance in PDAC. Inhibition of glycogen synthase kinase 3β (GSK3β) may offer a promising novel approach for modulating dynamic EMT plasticity to overcome resistance to FOLFIRINOX (5-Fluororuacil, Irinotecan, Oxaliplatin, FFX). Methods: Cell line panels comprising both epithelial (E) and quasi-mesenchymal (QM) subtypes were used and treated with IC50 (0.3125 mM) and IC100 (1.25 mM) doses of elraglusib (ELR). Gene expression patters in response to treatment were analyzed using bulk RNA sequencing. 6 patients in the safety run-in cohort of NCT05077800 treated with standard doses of FFX, ELR and losartan were included in this interim analysis. Biopsy specimens were processed for RNA-in situ hybridization (RNA-ISH), a clinically validated assay, and Nanostring (GeoMx) spatial transcriptomics. Results: In preclinical models, exposure to the higher IC100 (1.25 mM) doses of ELR in the QM cell lines caused a shift in transcriptional expression patterns similar to the E cell line, with strong downregulation of EMT. FFX exposure alone in QM cell lines was associated with significant resistance, but addition of ELR (even at IC50 dose) caused a synergistic effect with decreased cell viability. However, in the E cell lines, there was no benefit from addition of ELR. In the safety cohort, clinical results are encouraging with a long-term (&amp;gt;6 month) response in 3/6 patients. 2/6 achieved partial response, 1/6 had stable disease. Nanostring (GeoMx) spatial transcriptomic profiling on available biopsy samples showed significant global gene expression changes before and after treatment. There were concordant changes in 97 differentially expressed genes (DEGs) within the epithelial compartment with treatment, similar to ELR induced changes in PDAC cell lines, with a shift in transcriptional expression patterns from QM to E subtype. Gene set enrichment analysis demonstrated downregulation of the EMT pathway, as well as decreased TGF-β signaling. RNA-ISH analysis on baseline biopsy samples showed that the two partial (best) responders had higher degrees of QM marker expression (82.58% and 63.76%). The regimen was well tolerated with no dose limiting toxicity noted in the safety cohort. Conclusion: Our experiments demonstrate that the E subtype PDAC cell lines are inherently sensitive to FFX chemotherapy with no added advantage with GSK3β inhibition. Meanwhile, GSK3β inhibition in QM cell lines induces mesenchymal to epithelial transition, thereby rendering it sensitive to FFX. The data from our safety cohort provide a clinical proof of concept that GSK-3β inhibition with ELR, in combination with FFX, may be an effective therapeutic strategy in metastatic PDAC. NCT05077800 is currently enrolling. Citation Format: Priyadarshini Pathak, Eric Lin, Ildiko Phillips, David Ting, Colin Weekes. FOLFIRINOX with Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitor Elraglusib and Transforming Growth Factor- β (TGFβ) Inhibitor Losartan in Untreated Metastatic Pancreatic Ductal Adenocarcinoma(PDAC): Interim analysis of safety cohort [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C073.

Attainment of EULAR/ERA-EDTA targets of therapy with current immunosuppressive regimens and adjustments in treatment: a multicentre, real-life observational study

ObjectiveTo estimate real-life European Alliance of Associations for Rheumatology (EULAR)/European Renal Association (ERA)-European Dialysis and Transplantation Association (EDTA) response rates and predictors for no response in patients with lupus nephritis...

A Pilot Study of the CD38 Antagonist Daratumumab in Patients with Metastatic Renal Cell Carcinoma or Muscle-Invasive Bladder Cancer.

We performed a pilot study of daratumumab (an mAb directed against CD38) in muscle-invasive bladder cancer (MIBC) and treatment-refractory metastatic renal cell carcinoma (mRCC). Patients with MIBC underwent baseline transurethral resection of the bladder tumor followed by four weekly doses of daratumumab prior to cystectomy. Patients with mRCC underwent baseline and sequential biopsies after eight weekly doses. The primary endpoint was safety. The secondary endpoints were pathologic complete response rate for the MIBC cohort and objective response rate and progression-free survival for the mRCC cohort. Exploratory analyses included immune monitoring and overall survival. A Bayesian sequential monitoring design for toxicity was used for excessive toxicity. In both the MIBC (n = 8) and mRCC (n = 8) cohorts, no toxicity events were encountered. In the MIBC cohort, one patient experienced pathologic complete response rate. In the mRCC cohort, no objective responses were reported, and the median progression-free survival was 1.5 months (95% confidence interval, 1.1-1.8 months). Immune monitoring found significant reductions in NK cells in circulation in both cohorts after treatment. In the tissue analysis, IHC found evidence of diminished CD38 presence in mRCC with treatment, whereas the baseline levels in MIBC were low. Treatment with daratumumab was safe. No signal of efficacy was detected in mRCC, and conclusions on the activity in MIBC were limited. Evidence of daratumumab targeting CD38 was detected in circulating immune cells and within the tumor microenvironment of mRCC and MIBC. In this prospective clinical trial of daratumumab, treatment in patients with MIBC and mRCC was safe. Limited efficacy was observed. Treatment with daratumumab resulted in CD38-expressing immune cell subsets to be targeted both in circulation and within the tumor microenvironment.

Tumor immune microenvironmental characteristics in Human Epidermal Growth Factor-2 (HER2) positive esophageal adenocarcinoma: A comparative analysis and biomarker study

BackgroundHER2 targeting in esophageal adenocarcinoma (EAC) has shown potential, but often fails to show durable response. Given the contributions of the tumor immune microenvironment (TIME) to therapeutic responses, we aimed to chart the TIME characteristics of HER2 positive tumors. Methods84 biopsies were taken from the TRAP cohort (neoadjuvant chemoradiotherapy (nCRT) according to CROSS with trastuzumab and pertuzumab; n = 40; HER2+n = 40) and a control cohort with nCRT only (n = 44; HER2- n = 40, HER2+n = 4) before treatment. Biopsies were analysed using targeted gene expression analysis (Nanostring immune-oncology panel, 750 genes). Differential gene expression was assessed between HER2 positive (n = 44) vs. negative biopsies (n = 40), and non-responders (n = 17) vs. responders (n = 23) to anti-HER2 treatment. Statistical significance was determined as p-value <0.05, adjusted for multiple testing correction. Results83 biopsies were eligible for analyses following quality control (TRAP cohort n = 40; control cohort n = 43); there were no significant differences in clinical characteristics between the TRAP vs. control the cohort or HER2 positive vs. HER2 negative biopsies. HER2 expression was found to associate with epithelial markers (EPCAM p < 0.001; E-cadherin p < 0.001). Moreover, HER2 expression was associated with a lower expression of immune cell infiltration, such as NK-cells (p < 0.001) and CD8 T-cells (p < 0.001), but also lower expression of immune exhaustion markers (PDCD1LG2, CTLA4; p < 0.001). In non-responders to anti-HER2 treatment, baseline biopsies showed increased expression of immune exhaustion markers, as well as hypoxia and VEGF signalling. DiscussionHER2 expression was associated with epithelial tumor characteristics. The HER2 positive TIME showed reduced immune cell infiltration but also lower expression of inhibitory signals associated with immune exhaustion, questioning the mechanism behind potential clinical benefit of co-administration of anti-HER2 agents and checkpoint inhibitors. As limited response was associated with increased VEGF signalling, studies could investigate potential synergism of targeting VEGF and HER2.

75 WIRE: Window of Opportunity Clinical Trials Platform for Evaluation of Novel Treatments Strategies in Renal Cell Cancer

Abstract Background Despite the success of immunotherapy and VEGFR directed tyrosine kinase inhibitors (TKIs) for the treatment of renal cell carcinoma (RCC), a significant proportion of patients display primary or secondary resistance to systemic therapy. New treatment approaches are needed for these patients. Pre-surgical clinical trials allow us to study the effects of cancer treatment on the tumour microenvironment, comparing baseline biopsy with post-treatment nephrectomy in a ‘window of opportunity’ study. The results may give us insight into the mechanisms of new cancer therapies, that may be advanced into further clinical trials. Methods The WIRE trial (NCT03741426) is a phase II, multi-centre, multi-arm, non-randomised, neoadjuvant clinical trial platform. The overall aim of the trial is proof of mechanism for new RCC therapies. There are five investigational arms: 1. Cediranib (a VEGFR directed TKI), 2. Cediranib + Olaparib (a PARP inhibitor), 3. Olaparib, 4. Volrustomig (anti PD-1/CTLA-4 bispecific) and 5. Rilvegostomig (anti PD-1/TIGIT bispecific). The study uses a Bayesian adaptive design to optimise recruitment, with pre-specified criteria assessed at interim analyses to stop for futility, continue recruitment to that arm, or move to the next investigational arm. Eligible patients have cT1b+, cN0/1, cM0/1 clear cell RCC, planning to undergo surgery, without medical contraindication to trial therapy. Patients undergo baseline biopsy to confirm clear cell RCC and multiparametric dynamic contrast enhanced (DCE) MRI scan of the primary tumour. For tablet arms 1-3, patients receive at least 14 days of investigational medicinal product (IMP). For immunotherapy arms 4 &amp; 5, patients receive one infusion of IMP. The tumour is re-evaluated by DCE-MRI immediately before surgery. At nephrectomy, multi-region tissue sampling from the tumour is performed both pre-arterial ligation and post resection. Serial translational blood and urine samples are collected before, during and after treatment. The primary endpoint for arms 1-3 is a ≥30% reduction in DCE-MRI assessed capillary permeability (Ktrans) compared to baseline. The primary endpoint for arms 4 &amp; 5 is a ≥30% increase in IHC assessed CD8+ T cell density comparing the resected tumour to baseline biopsy. Secondary endpoints include RECIST v1.1 primary tumour response and adverse events. A comprehensive translational science programme accompanies the trial, which will assess the tumour microenvironment and peripheral blood response to therapy, using techniques including imaging mass cytometry and single cell RNA sequencing. This will allow us to gain a deep understanding of mechanisms of response in each investigational arm. To date, arms 1 &amp; 2 have completed recruitment, and arm 3 is actively recruiting. Conclusions WIRE offers a unique opportunity to investigate the mechanisms of new therapies for RCC. Successful completion of these peri-surgical studies depends on co-ordination between many specialties, including surgery, oncology, radiology, and pathology. The adaptive design allows for efficient allocation of patients to treatment arms to generate optimal data for each IMP. WIRE may identify novel biomarkers of response and toxicity, to inform treatment selection for patients. The data generated will be a foundation for further trials of these IMPs in advanced disease.

Immune Modulation in Untreated, Contralateral Hepatic Metastases after Yttrium-90 Radioembolization of Microsatellite Stable Colorectal Cancer

PurposeTo assess immunogenic effects in unembolized contralateral tumor after single lobar yttrium-90 transarterial radioembolization (90Y-TARE) of colorectal liver metastases (CRLMs). Material and MethodsThe analysis comprised 10 patients with microsatellite stable (MSS) CRLM scheduled for staged treatment in the prospective AROMA trial. Eligibility criteria included bilobar metastatic disease with >5 lesions without any treatment within 3 weeks. Baseline biopsy was followed by initial 90Y-TARE treatment of 1 liver lobe, followed by a second biopsy of yet untreated tumors in the other liver lobe at a median of 13 days (range, 4–49 days) immediately before second treatment. Tumor biopsies and peripheral blood mononuclear cells (PBMCs) were collected before treatments for immune cell analysis. Patients were stratified into responders and nonresponders based on tumor control or progression during follow-up. ResultsAt baseline, responders (n = 4) displayed lower concentrations of FoxP3+ cells and colocation of CD4+FoxP3+ cells than nonresponders (both P = .02) in tumor tissues. At second biopsy, nonresponders showed a higher CD68+ macrophage density (P = .0014) than responders. Responders displayed fewer CD4+FoxP3+ T cells than CD8+ T cells at all time points (P = .02 and P = .0428). Nonresponders demonstrated a trending increase in CD68+ macrophages (P = .062), as well as a higher CD8+PD1+/CD8+ ratio (P = .062). PBMCs of nonresponders displayed lower CD8+PD1+ T cells and CD8+PD1+/CD8+ ratio at both time points. Conclusions90Y-TARE induces local immunogenic effects in nonexposed MSS CRLM, as well as systemic exhaustion of immune cells in nonresponders. Clinical implications such as a prognostic role or synergism of 90Y-TARE and checkpoint inhibition in MSS CRLM warrant further investigation.

SACI-IO HR+: A randomized phase II trial of sacituzumab govitecan with or without pembrolizumab in patients with metastatic hormone receptor-positive/HER2-negative breast cancer.

LBA1004 Background: Sacituzumab govitecan (SG) is a TROP2 antibody drug conjugate (ADC) with a topoisomerase I inhibitor payload (SN-38) approved for previously treated triple negative and hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC). Double-strand DNA breaks induced by SN-38 activate cGAS-STING, stimulating type I IFN production and T cell recruitment. SN-38 can upregulate MHC class-I and PD-L1 expression, enhance cytotoxic T cell effector functions and deplete regulatory T cells. To evaluate if SG synergizes with pembrolizumab (PD-1 inhibitor) we conducted a randomized, open-label phase 2 study comparing SG with or without pembrolizumab in HR+/HER2- MBC (NCT04448886). Methods: Eligible patients (pts) had unresectable locally advanced or metastatic HR+ (ER≥1% and/or PR≥1%), HER2- breast cancer treated with ≥1 prior endocrine therapy and 0-1 chemotherapy (CT) for MBC. Pts with brain metastases were eligible if locoregional therapy was completed and steroids discontinued ≥7 days before study therapy. Pts who received prior topoisomerase I inhibitor ADC, irinotecan or PD-1/L1 inhibitors were excluded. Pts were randomized 1:1 to Arm A [SG 10 mg/kg IV (D1, D8) + pembrolizumab 200 mg IV (D1), 21-day cycle] or Arm B (SG alone). The primary endpoint was progression-free survival (PFS); secondary endpoints included PFS in PD-L1+ pts (22C3 CPS ≥1), overall survival (OS), objective response rate (ORR) and toxicity (NCI CTCAE v5.0). Baseline and on-treatment biopsies were performed for correlative analyses. For this preliminary analysis, data were locked 1/12/24. Results: Between 03/2021-01/2024, 110 pts enrolled; 104 pts (52 Arm A; 52 Arm B) started study therapy and were included in the analysis. Median age was 57 yrs (range: 27-81); 102 pts (98.1%) were female. 80 pts (76.9%) received prior CDK4/6 inhibitor for MBC; 58 (55.8%) had no prior CT, 46 (44.2%) had 1 prior line of CT for MBC. At a median follow-up of 9.2 months (mo), median PFS was 8.4 mo in Arm A vs 6.2 mo in Arm B (HR 0.76, 95% CI 0.47-1.23, log-rank p=0.26); ORR 21.2% and 17.3%, respectively. OS data are immature with only 26 events to date; OS was 16.9 mo vs 17.1 mo (HR 0.65, 95% CI 0.30-1.41, log-rank p=0.28), respectively. The most frequent treatment-related toxicities (≥G2) in Arm A were neutropenia (67.3%), fatigue (36.5%), alopecia (36.5%), anemia (32.7%), leukopenia (26.9%), diarrhea (21.2%) and nausea (21.2%); in Arm B, neutropenia (59.6%), alopecia (38.5%), diarrhea (34.6%), nausea (32.7%), fatigue (32.7%) and anemia (21.2%). Conclusions: Addition of pembrolizumab to SG showed a non-significant trend toward improved PFS in unselected HR+/HER2- MBC at this preliminary time point. Final PFS and updated OS with further follow-up will be presented at the meeting. Exploratory outcome analyses by TROP2 and PD-L1 expression will be reported. Clinical trial information: NCT04448886 .

A phase 2 study evaluating response and biomarkers in patients with microsatellite stable (MSS) advanced colorectal cancer (CRC) treated with nivolumab/relatlimab.

3554 Background: Lymphocyte-activation gene 3 (LAG-3) is an immunosuppressive signal, impairing T effector (Teff) activation. Pre-clinical studies suggest LAG-3 inhibition promotes anti-tumor immunity by enhancing Teff function. Correlative studies linked increased LAG-3 levels with reduced overall survival (OS) in CRC. These data prompted optimism that combined LAG-3/PD-1 inhibition might promote anti-tumor responses in MSS CRC. Methods: We conducted a phase 2 study of nivolumab (Nivo) (aPD1)/relatlimab (Rela) (aLAG3) in patients with previously treated advanced MSS CRC. This study enrolled patients onto 1 of 3 arms. Arms A and B used a Composite PD-L1 and acellular Mucin (CPM) score to define patients as CPM(+) in Arm A (CPM≥ 15%) and CPM(-) in Arm B (CPM&lt; 15%). Patients in Arm A and B received Nivo 480mg/Rela 160mg IV q4 weeks. Arm C enrolled patients without biomarker selection who received Nivo 480mg/Rela 960mg or Rela 480mg/Nivo 480mg. The primary endpoint was objective response, and each arm was powered to detect an objective response rate (ORR) of ≥20%. Results: Fifty nine evaluable patients were enrolled (Arm A: 12, Arm B: 15, Arm C: 32). Patient characteristics and responses are outlined. Treatment was well tolerated with no new safety signals over aPD1 alone. Three (5%) patients achieved a partial response (PR), 6 (10%) stable disease (SD), and 50 with progression of disease (PD) as best response. The median progression-free survival was (Arm A: 2.2 mo., Arm B: 2.8 mo., Arm C: 2.6 mo.) while median OS was (Arm A: 4.3 mo., Arm B: 14.2 mo., Arm C: 12.4 mo.). In N=6 patients with lung-only metastases, 2 (33%) experienced a PR, and 1 (17%) a SD, for a clinical benefit rate of 50%. The trial incorporated translational correlates. We will report mass cytometry on PBMCs, RNAseq on baseline biopsies, and imaging mass cytometry on pre- and on-treatment biopsies. Conclusions: This combination did not meet the primary endpoint of 20% ORR for any of the 3 cohorts, but there was intriguing possible activity in patients with lung only metastases, consistent with emerging data. Clinical trial information: NCT03642067 .

A phase I trial of intratumoral STX-001: A novel self-replicating mRNA expressing IL-12 alone or with pembrolizumab in advanced solid tumors.

TPS2696 Background: STX-001 is a multi-mechanistic lipid nanoparticle encapsulated synthetic self-replicating mRNA, engineered to express the IL-12 cytokine for an extended duration when administered intratumorally. A murine surrogate of STX-001 induces deep durable responses in multiple preclinical solid tumor models. This Phase 1 open-label, multi-center first-in-human dose-escalation trial evaluates the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of STX-001 alone, or in combination with pembrolizumab in patients with treatment refractory advanced cancers. Methods: Approximately 30 adults ≥18 years of age with histologically confirmed advanced solid tumors that are refractory to standard treatments will be enrolled. The study will consist of two arms, an STX-001 monotherapy arm and an STX-001 + pembrolizumab combination arm, enrolling approximately 15 patients each. Key eligibility criteria include ECOG status 0 or 1, a tumor lesion amendable to injection, and confirmed prior disease progression. STX-001 will be administered every 3 weeks (q3w) for 3 doses and then every 6 weeks (q6w) for a further 3 doses. Tumor biopsies will be obtained at baseline and after the first STX-001 administration. Primary endpoints will include safety and maximum tolerated dose (MTD), with secondary endpoints encompassing pharmacokinetics and preliminary efficacy as measured by RECIST 1.1. To determine the MTD, this study will employ the Bayesian optimal interval (BOIN) design, with the 3 + 3 design run-in and a recommended phase 2 dose (RP2D) will be determined. Tumor response will be assessed using RECIST 1.1 criteria. Exploratory pharmacodynamic analyses will assess tumor-infiltrating lymphocytes and STX-001 mRNA in baseline and on-treatment biopsies, evaluate inflammatory cytokine profiles in plasma, determine immune activation in circulating immune cells, and measure circulating tumor DNA (ctDNA) for molecular response assessment. Clinical trial information: NCT06249048 .

EVICTION study: ICT01, an anti-Butyrophilin 3A monoclonal antibody activating γ9δ2 T cells in combination with pembrolizumab in checkpoint inhibitor refractory melanoma.

9534 Background: γ9δ2 T-cell tumor infiltration is associated with a favorable prognosis making these cells a new potential target for immunotherapy. The anti-BTN3A mAb ICT01 selectively activates γ9δ2 T cells and is being studied in the phase1/2a EVICTION Trial (NCT04243499). In preclinical studies, ICT01 induces upregulation of PD-1 on γ9δ2 T-cells and combination with pembrolizumab leads to enhanced cancer cell killing, providing scientific rationale to evaluate this combination. Here we present interim results from EVICTION of ICT01 in combination with pembrolizumab in patients with checkpoint inhibitor (CPI) refractory melanoma. Methods: EVICTION evaluated ICT01 (20 µg to 200 mg, Q3W) plus pembrolizumab (200mg IV Q3W) in a dose escalation solid tumor basket design that resulted in an ongoing expansion cohort of patients with CPI refractory melanoma (2 dose levels, 7 and 200 mg ICT01). Patients are selected based on higher baseline γ9δ2 T cells. Efficacy evaluations by i/RECIST 1.1 are conducted every 8 weeks. Disease Control Rate (DCR) as primary efficacy endpoint is defined as the sum of complete response (CR), partial response (PR) and stable disease (SD, minimum week 16). Baseline and on-treatment blood and biopsy specimens are collected for correlative translational work. Results: ICT01 plus pembrolizumab has a favorable safety profile with first-dose Grade 1/2 infusion related reactions (IRR) and cytokine release syndrome (CRS) as most common adverse events (in 38% and 19% of patients respectively, 5%and 2% Grade 3) among all doses and indications. To date, 35 CPI refractory melanoma patients have been enrolled, of which 65% have received 2+ prior lines of CPI. Currently 21 patients are evaluable at week 16 with a DCR of 42% (including 3 PR) and a 6-month progression free survival of 38%. In the circulation, full BTN3A receptor occupancy on immune cells was achieved at 7 mg dose, with down-modulation of BTN3A observed at doses greater than 20 mg. Clinical response was related to baseline tumoral BTN3A expression, sustained elevation of IFNg levels, and tumor microenvironment remodeling, including increased PD-L1 expression and CD8 T cells proliferation and activation. Conclusions: ICT01 in combination with pembrolizumab has a favorable safety profile and promising efficacy data. Patient selection based on BTN3A tumor expression will be further evaluated as an enrichment strategy. Clinical trial information: NCT04243499 .

RNA-Seq based gene expression profiling of baseline and on-treatment breast tumors to predict response to HER2-directed therapy, without chemotherapy (TBCRC026).

530 Background: Robust biomarkers are needed to personalize treatment in early stage HER2-positive breast cancer. The TBCRC026 trial (n=83) demonstrated that early metabolic changes on FDG-PET/CT [&gt;40% decline in SULmax at day 15 post treatment initiation (C1D15); C1D15 SULmax] were associated with response to neoadjuvant trastuzumab/pertuzumab (HP), without chemotherapy, in an ER-negative, HER2-positive cohort (NCT01937117). We investigated the association of RNA sequencing (RNAseq)-based gene expression signatures (GES) at baseline and on-treatment (C1D15), with early metabolic change on PET/CT and clinical outcomes. Methods: RNAseq (664 GES) was performed on baseline (n=56) and C1D15 (n=31) tumor biopsies (25 paired samples available). Association with pCR following HP (secondary), PET/CT parameters and survival (exploratory) was studied. Gene expression changes between baseline and C1D15 samples were tested by Wilcoxon signed-rank test. Association between GES and PET/CT parameters and GES with pCR were evaluated using logistic regression models. Cox regressions were performed to study the association between PET/CT SULmax at C1D15 and a B cell signature and relapse-free survival (RFS). The likelihood ratio test was used to compare the prognostic ability of two nested Cox models. Results: Both RNAseq and clinical outcomes were available in 53/83 patients. HER2-enriched (HER2-E) intrinsic subtype comprised 71% (40/56) of cases at baseline and 61% (19/31) at D15; basal subtype for 27% (15/56) and 39% (12/31), respectively. pCR rate was 19% (10/53) with all but one exhibiting HER2-E subtype. One basal-like sample underwent pCR, with downregulation of HER2 amplicon and IgG signatures slightly more prominent. Gene expression changes from baseline to C1D15 were analyzed; ERBB2was downregulated after HP (p = 0.007), B cell signatures, CD4, CD8, natural killer cells and Treg cells were significantly upregulated at C1D15. GES associated with 40% decline in SULmax at C1D15 included ERBB2 amplicon genes ( ERBB2, GRB7) and correlation to the HER2-E subtype, whereas basal-like signatures were significantly associated with lack of response on PET at C1D15. Univariate logistic regression analysis suggested that FOS-JUN, fibroblast and B cell GES at baseline were significantly associated with pCR (p=&lt;0.05). Finally, the addition of a B cell signature improved the prognostic value of C1D15 SULmax on PET/CT for RFS (p &lt; 0.05). Conclusions: In this unique cohort with ER-negative, HER2-positive breast cancer treated with neoadjuvant HP without chemotherapy, HER2-E tumors and those with increased immune signaling were more likely to respond to HER2-directed therapy. Combining C1D15 SULmax on PET/CT and a B cell signature provided improved prognostic information and should be further explored as candidate biomarkers in this setting. Clinical trial information: NCT01937117 .

Safety, efficacy, and biomarker results from an open-label, multicenter, phase 1 study of RP2 alone or combined with nivolumab in a cohort of patients with uveal melanoma.

9511 Background: Uveal melanoma is the most common primary intraocular tumor in adults and has a high risk of liver metastasis. Effective treatments for metastatic uveal melanoma (MUM) are limited, as MUM responds poorly to immune checkpoint inhibition. RP2 is an enhanced-potency oncolytic HSV-1–expressing human GM-CSF, a fusogenic glycoprotein (GALV-GP-R−), and an anti–CTLA-4 antibody-like molecule. We present safety, efficacy, and biomarker data for RP2 monotherapy and RP2 +nivolumab (nivo; anti–PD-1) in patients (pts) with MUM (NCT04336241). Methods: Pts ≥18 years old with advanced or metastatic non-neurologic solid tumors (including MUM) who progressed on, or could not tolerate, standard therapy were included in the clinical trial; pts had ≥1 measurable and injectable tumor (≥1 cm). After determination of the recommended phase 2 dose of intratumoral RP2 (1×106 PFU/mL once, then ≤7 doses at 1×107 PFU/mL; ≤10 mL total/treatment day), pts received RP2 Q2W as monotherapy or in addition to nivo (240 mg Q2W/480 mg Q4W). Responses were assessed per modified RECIST v1.1.Tumor biopsies and peripheral blood mononuclear cells were collected pre-treatment and at day 43 and were analyzed by immunohistochemistry (IHC) and/or sequencing of the CDR3β region of the T-cell receptor (TCR) by immunoSEQ. Results: A total of 17 pts with MUM were enrolled (RP2 monotherapy, n = 3; RP2 + nivo, n = 14). Most pts had received both anti–PD-1 and anti–CTLA-4 therapy (12/17 [70.6%]), and 3/17 (17.6%) had received ≥3 prior lines of therapy. The ORR was 29.4% (5/17; all PRs; RP2 monotherapy, 1/3; RP2 + nivo, 4/14). The DCR (CR + PR + SD) was 58.8% (10/17). The median (range) duration of response at the data cutoff was 11.5 (2.8–21.2) months, with some responses and disease stabilizations ongoing. The most common overall grade 1–2 treatment-related AEs (TRAEs; ≥20% overall) were pyrexia, chills, fatigue, hypotension, and pruritus. The only grade 3 TRAE in &gt; 1 pt was hypotension (2 pts receiving RP2 + nivo); no grade 4/5 TRAEs occurred. Evaluable baseline and post-treatment biopsies were used for IHC (n = 8) and TCR sequencing analyses. Paired biopsies from pts with clinical benefit (n = 5; 2 PR, 3 SD) showed an increase in tumor PD-L1 expression by IHC analysis, and 4 of these pts (2 PR, 2 SD) exhibited an increase in CD8+ T-cell infiltration into tumors. TCR sequencing following treatment with RP2 + nivo revealed expansion of pre-existing TCRs and generation of new T-cell clones. Conclusions: RP2 alone or combined with nivo demonstrated a favorable safety profile and meaningful antitumor activity in pts with previously treated MUM. Biomarker data indicated immune cell infiltration and increased PD-L1 expression in tumors and changes in the peripheral T-cell repertoire following RP2 ± nivo therapy. Based on these results, a randomized, controlled clinical development plan is being planned. Clinical trial information: NCT04336241 .

Cost-effectiveness of a circulating tumor fraction molecular biomarker for treatment response monitoring.

11082 Background: Clinical validation studies have demonstrated that molecular biomarkers quantifying ctDNA changes in circulating tumor fraction (TF) predict survival outcomes and may be used for treatment response monitoring (TRM). While clinical utility studies to determine the impact on outcomes of molecular biomarker-driven treatment decisions versus standard of care imaging are ongoing, cost-effectiveness has not been evaluated. Here we evaluate the cost-effectiveness of a molecular biomarker, Tempus xM, used for TRM. Methods: xM quantifies changes in TF from baseline and on-treatment liquid biopsies. We use a patient-level Markov simulation to compare xM-guided treatment (intervention) to diagnostic imaging-guided treatment (control) over 24 weeks of therapy. In both arms xM and imaging is assessed at 12 weeks and treatment decisions are made based on xM (intervention) or diagnostic imaging (control). Imaging and xM concordance (Table) was based on a retrospective, real-world (RW) study of 51 patients tested with xM that received rw-imaging that evaluated the association of xM with rw-outcomes in advanced pan-cancer patients treated with immune checkpoint inhibitors (ICIs) +/- chemotherapy (CT). We assume non-responders discontinue ICIs and switch to CT and responders remain on ICIs. Appropriate (inappropriate) therapy was defined as treatment decisions concordant (discordant) with xM results. Costs were calculated from Medicare’s perspective in 2023 USD. Control patients do not accrue the cost of xM. Sensitivity analyses were conducted. Results: Intervention patients saved $4,400 ($63,424 vs. $67,824 in controls) by preventing use of ineffective therapy (4.3 weeks avoided vs 0.2 weeks in controls), a savings of $1076 per week of ineffective therapy avoided. In all sensitivity analyses, xM-guided therapy remained cost effective (Table). The timing of therapy intervention and the cost of therapy had the largest impact on savings. Conclusions: This model demonstrates that xM-guided treatment is cost-saving compared to imaging alone during 24 weeks of treatment. Future work will incorporate variable clinical uptake and long-term outcomes by expanding the time horizon and including treatment discontinuation due to toxicities and mortality. [Table: see text]

Investigation of a potential protein biomarker signature that may predict clinical benefit of NT-I7 and pembrolizumab in patients with cold gastrointestinal tumors.

2563 Background: Microsatellite stable colorectal (MSS-CRC) and pancreatic cancer (PDAC) are immunologically cold tumors due to low immunogenicity and lack of genomic diversity. NT-I7, a long-acting IL-7, and pembrolizumab (pembro) show efficacy in these hard-to-treat indications. While a limited set of patients (pts) achieve objective response, frequency and duration of the disease control rate point to a larger subset obtaining clinical benefit. To identify novel predictive biomarkers, we analyzed baseline peripheral and biopsy samples from pts based on treatment duration. Methods: Open-label Phase 2a study in pts with relapsed/refractory checkpoint inhibitor-naïve MSS-CRC and PDAC; NT-I7 1200 µg/kg IM every 6 weeks (Q6W), pembro 200 mg IV Q3W. Subjects were grouped by treatment duration, measured in NT-I7 doses administered before treatment discontinuation for any cause: 1 dose was short (ST), 2-3 doses medium (MT) and ≥4 doses long (LT). Correlative studies included peripheral (proteomics, T cell receptor sequencing [TCRseq], single cell RNA sequencing [scRNAseq]), and biopsy (genomics, transcriptomics, TCRseq). Results: As of 02OCT2023, 53 evaluable pts completed or discontinued treatment; 5 are still on follow up. ST group included 21 pts, MT 22 and LT 10 (including all 5 partial responders). Tumor biopsies were confirmed MSS with low tumor mutational burden (TMB). LT pts had similar age (59.0 [53.0-71.5] vs 66.0 [47.3-73.5]; ST vs LT) and lower baseline tumor burden (81.0 mm vs 58.0 mm; ST vs LT, p = 0.022). Biopsy analysis showed LT pts had, at baseline, upregulated pathways related to immune activity despite confirmed cold tumor status. Baseline scRNAseq in peripheral blood showed that stem-like CD8 T cells (precursors of exhausted [TPEX] and stem-cell memory [TSCM]) had a differential activation pattern in LT pts; those pathways were enriched in memory effector subsets in ST pts. Preserved antigen-specific stemness may be needed for NT-I7 + pembro efficacy. Baseline concentrations of 3 proteins that can be produced by growing tumors were significantly increased in ST pts. Pts were classified based on elevated levels of these potential biomarkers: POSITIVE (≥2 biomarkers; 20 pts) and NEGATIVE (≤1 biomarker; 33 pts). Pts with NEGATIVE signature at baseline had significantly higher overall survival, regardless of ST, MT or LT status (13.2 vs 8.9 months, p = 0.030). Conclusions: Preserved tumor-specific TPEX activity may be required for NT-I7 + pembro activity based on its presence in LT pts, who remained on treatment the longest. According to this analysis, there are 3 potentially predictive protein biomarkers that may help identify a pt subset more likely to experience clinical benefits from the combination treatment of NT-I7 + pembro. Further verification of the predictive nature of this signature in independent cohorts is ongoing. Clinical trial information: NCT04332653 .