Non-responding Patients Research Articles

Impact of minimal residual disease response and of status of disease on survival after blinatumomab in B-cell acute lymphoblastic leukemia: results from a real-life study.

Blinatumomab is a bispecific T-cell engager approved for relapsed/refractory and minimal residual disease positive B-cell Acute Lymphoblastic Leukemia. We conducted a retrospective study evaluating the outcome of Blinatumomab. The impact of clinical and treatment-related variables on cumulative incidence of relapse/progression (CIRP), event-free (EFS) and overall survival (OS) was analyzed. From January 2016 to December 2022 50 Ph'- (37) and Ph+ (13) B-ALL patients received Blinatumomab. The median age was 37. Indications to blinatumomab were relapsed/refractory B-ALL in 29 and MRD-positive in 21 patients. Blinatumomab was the 2nd and 3rd line in 40 and in 10 patients, respectively. Twenty patients were treated pre-transplantation, ten were treated for relapse after transplant, twenty were not eligible for transplant. Out of 29 patients treated for relapsed/refractory disease, 16 (55%) achieved complete response and 12 achieved MRD-negativity. Out of 21 patients treated for MRD, 16 (76%) achieved MRD-negativity. At a median follow-up of 46 months the median EFS and OS were 11.5 and 16.2 months. The CIRP was 50%. In univariate analysis age, disease-status (overt vs. minimal disease) at blinatumomab, bridging to transplant after blinatumomab and MRD-response resulted significant for EFS and OS. In multivariate analysis only disease-status and MRD-response retained significance both for EFS and OS. Disease-status and MRD-response resulted significant for EFS and OS also after censoring at HSCT. This retrospective study on B-ALL patients treated with blinatumomab confirms a superior outcome for MRD-responsive over MRD non-responsive patients. Survival depends also on the disease-status prior treatment.

Targeting SMAD3 Improves Response to Oxaliplatin in Esophageal Adenocarcinoma Models by Impeding DNA Repair.

TGFβ signaling is implicated in the progression of most cancers, including esophageal adenocarcinoma (EAC). Emerging evidence indicates that TGFβ signaling is a key factor in the development of resistance toward cancer therapy. In this study, we developed patient-derived organoids and patient-derived xenograft models of EAC and performed bioinformatics analysis combined with functional genetics to investigate the role of SMAD family member 3 (SMAD3) in EAC resistance to oxaliplatin. Chemotherapy nonresponding patients showed enrichment of SMAD3 gene expression when compared with responders. In a randomized patient-derived xenograft experiment, SMAD3 inhibition in combination with oxaliplatin effectively diminished tumor burden by impeding DNA repair. SMAD3 interacted directly with protein phosphatase 2A (PP2A), a key regulator of the DNA damage repair protein ataxia telangiectasia mutated (ATM). SMAD3 inhibition diminished ATM phosphorylation by enhancing the binding of PP2A to ATM, causing excessive levels of DNA damage. Our results identify SMAD3 as a promising therapeutic target for future combination strategies for the treatment of patients with EAC.

Circulating immunome fingerprint in eosinophilic esophagitis is associated with clinical response to proton pump inhibitor treatment.

The aim of the study was to characterize the circulating immunome of patients with EoE before and after proton pump inhibitor (PPI) treatment in order to identify potential non-invasive biomarkers of treatment response. PBMCs from 19 healthy controls and 24 EoE patients were studied using a 39-plex spectral cytometry panel. The plasmacytoid dendritic cell (pDC) population was differentially characterized by spectral cytometry analysis and immunofluorescence assays in esophageal biopsies from 7 healthy controls and 13 EoE patients. Interestingly, EoE patients at baseline had lower levels of circulating pDC compared with controls. Before treatment, patients with EoE who responded to PPI therapy had higher levels of circulating pDC and classical monocytes, compared with non-responders. Moreover, following PPI therapy pDC levels were increased in all EoE patients, while normal levels were only restored in PPI-responding patients. Finally, circulating pDC levels inversely correlated with peak eosinophil count and pDC count in esophageal biopsies. The number of tissue pDCs significantly increased during active EoE, being even higher in non-responder patients when compared to responder patients pre-PPI. pDC levels decreased after PPI intake, being further restored almost to control levels in responder patients post-PPI. We hereby describe a unique immune fingerprint of EoE patients at diagnosis. Moreover, circulating pDC may be also used as a novel non-invasive biomarker to predict subsequent response to PPI treatment.

Abstract LB445: USP24 suppresses T cell anti-tumor activity by enhancing the stability of the PD-1 protein

Abstract Persistent programmed cell death-1 (PD-1)/PD-L1 inhibitory signaling disrupts T cell cytotoxic function, leading to T cell exhaustion—a phenomenon strongly associated with advanced cancer progression and immunotherapy failure. Deciphering the mechanisms behind dysregulated PD-1 expression and reactivating tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) is crucial. Our previous research established that tumor-associated macrophage-derived IL-6 promotes STAT3-dependent PD-1 transcriptional activity and immunotherapy resistance. The current study further explores whether IL-6 abundance in the TME contributes to PD-1 protein stability. Results indicate that IL-6 extended PD-1 protein half-life in T cells, suggesting its role in modulating PD-1 protein stability. Furthermore, analysis of the GEO database showed a positive correlation between the expression of ubiquitin-specific-processing protease (USP) family enzymes and PD-1 in non-responding patients receiving anti-PD-1 therapy. Knockdown experiments revealed that USP24 strongly downregulated PD-1 protein expression and stability. USP24 depletion reversed IL-6-induced PD-1 expression and stability, with USP24 colocalizing with PD-1 at the plasma membrane upon IL-6 stimulation. Immunoprecipitation-Western blot assays confirmed that USP24 directly interacted with PD-1 and countered E3 ligase c-Cbl-mediated ubiquitination. Depleting USP24 suppressed PD-1-mediated immunosuppression both in vitro and in vivo. In lung-specific EGFRL858R/USP24C1698A functional knockout mice, tumor progression was slower, and exhausted tumor-infiltrating PD-1+ TIM-3+ CD8+ T cells reduced compared to EGFRL858R mice. Inhibiting PD-1 stability with an IL-6 neutralizing antibody or the novel inhibitor USP24-i-101 decreased tumor volume and increased activated cytotoxic T cell infiltration in vivo. Moreover, USP24-i-101 enhanced therapeutic effects in combination with anti-CTLA4 immunotherapy. Clinically, our in-house cohort revealed elevated USP24 expression in tumor-infiltrating lymphocytes or peripheral T cells, correlating with worse clinical outcomes and unfavorable responses to immunotherapy in lung cancer patients. This study uncovers USP24 as a novel PD-1 regulator, deubiquitinating PD-1 to enhance protein stability and suppress T cell function. In conclusion, USP24 emerges as a potential target for enhancing anti-tumor immunity and as a biomarker predicting clinical responses to immunotherapy in lung cancer. Citation Format: Hung-Chia Hsieh, Jan-Jong Hung, Yi-Ching Wang. USP24 suppresses T cell anti-tumor activity by enhancing the stability of the PD-1 protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB445.

CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line

BackgroundImmune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers.MethodsThe levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients).ResultsIn both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors.ConclusionHigh levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Cardiac hemodynamics phenotypes and individual responses to training in coronary heart disease patients.

In patients with coronary heart disease (CHD), individualized exercise training (ET) programs are strongly recommended to optimize peak oxygen uptake ( O2peak) improvement and prognosis. However, the cardiac hemodynamic factors responsible for a positive response to training remain unclear. The aim of this study was to compare cardiac hemodynamic changes after an ET program in responder (R) versus non-responder (NR) CHD patients. A total of 72 CHD patients completed a 3-month ET program and were assessed by cycle ergometer cardiopulmonary exercise test (CPET: O2peak assessment) with impedance cardiography (ICG) for hemodynamic measurements before and after training. Cardiac hemodynamics (e.g., CO, CI, SV, ESV, EDV, and SVR) were measured by ICG during CPET. The R and NR groups were classified using the median change in O2peak (>the median for R and ≤the median for NR). In the R group, O2peak (+17%, p < 0.001), CO, CI, SV, and HR increased by 17%, 17%, 13%, and 5%, respectively (p < 0.05) after the training program. In the NR group, O2peak, CO, CI, and SV increased by 0.5%, 5%, 8%, and 6%, respectively (p < 0.01). The SVR decreased in both groups (-19% in R and -11% in NR, p < 0.001). Among CHD patients, the R group showed a better improvement in peak cardiac output via an increase in peak stroke volume and heart rate and a reduced systemic vascular resistance than the NR group. Different cardiac phenotype adaptations and clinical individual responses were identified in CHD patients according to the aerobic fitness responder's status.

Total metabolic tumor volume on 18F-FDG PET/CT is a game-changer for patients with metastatic lung cancer treated with immunotherapy

PurposeBecause of atypical response imaging patterns in patients with metastatic non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICPIs), new biomarkers are needed for a better monitoring of...

Immunological Mechanisms behind Anti-PD-1/PD-L1 Immune Checkpoint Blockade: Intratumoral Reinvigoration or Systemic Induction?

Anti-PD-1/PD-L1 immune checkpoint blockade (ICB) has been widely used to treat many types of cancer. It is well established that PD-L1 expressing cancer cells could directly inhibit the cytotoxicity of PD-1+ T cells via PD-L1-PD-1 interaction. However, histological quantification of intratumoral PD-L1 expression provides limited predictive value and PD-L1 negative patients could still benefit from ICB treatment. Therefore, the current major clinical challenges are low objective response rate and unclear immunological mechanisms behind responding vs. non-responding patients. Here, we review recent studies highlighting the importance of longitudinal pre- and post-ICB treatment on patients with various types of solid tumor to elucidate the mechanisms behind ICB treatment. On one hand, ICB induces changes in the tumor microenvironment by reinvigorating intratumoral PD-1+ exhausted T cells ("releasing the brakes"). On the other hand, ICB can also affect systemic antitumor immunity in the tumor-draining lymph node to induce priming/activation of cancer specific T cells, which is evident by T cell clonal expansion/replacement in peripheral blood. These studies reveal that ICB treatment not only acts on the tumor microenvironment ("battlefield") but also acts on immune organs ("training camp") of patients with solid tumors. A deeper understanding of the immunological mechanisms behind ICB treatment will pave the way for further improvements in clinical response.

Psychomotor retardation: What about the partial responders to magnetic transcranial stimulation in treatment resistant depression ?

ObjectivePsychomotor retardation is a core clinical component of Major Depressive Disorder responsible for disability and is known as a treatment response marker of biological treatments for depression. Our objective was to describe cognitive and motoric measures changes during a treatment by repetitive Transcranial Magnetic Stimulation (rTMS) within the THETAD-DEP trial for treatment-resistant depression (TRD), and compare those performances at the end of treatment and one month after between responders (>50% improvement on MADRS score), partial responders (25–50%) and non-reponders (no clinically relevant improvement). Our secondary aim was to investigate baseline psychomotor performances associated with non-response and response even partial. MethodsFifty-four patients with treatment-resistant unipolar depression and treated by either high frequency 10 Hz rTMS or iTBS for 4 weeks (20 sessions) underwent assessment including French Retardation Rating Scale for Depression (ERD), Verbal Fluency test, and Trail Making Test A. before, just after treatment and one month later. Results20 patients were responders (R, 21 partial responders (PR) and 13 non-responders (NR). rTMS treatment improved psychomotor performances in the R and PR groups unlike NR patients whose psychomotor performance was not enhanced by treatment. At baseline, participants, later identified as partial responders, showed significantly higher performances than non-responders. ConclusionHigher cognitivo-motor performances at baseline may be associated with clinical improvement after rTMS treatment. This work highlights the value of objective psychomotor testing for the identification of rTMS responders and partial responders, and thus may be useful for patient selection and protocol individualization such as treatment continuation for early partial responders.

Multi-omics staging of locally advanced rectal cancer predicts treatment response: a pilot study

Treatment response assessment of rectal cancer patients is a critical component of personalized cancer care and it allows to identify suitable candidates for organ-preserving strategies. This pilot study employed a novel multi-omics approach combining MRI-based radiomic features and untargeted metabolomics to infer treatment response at staging. The metabolic signature highlighted how tumor cell viability is predictively down-regulated, while the response to oxidative stress was up-regulated in responder patients, showing significantly reduced oxoproline values at baseline compared to non-responder patients (p-value < 10–4). Tumors with a high degree of texture homogeneity, as assessed by radiomics, were more likely to achieve a major pathological response (p-value < 10–3). A machine learning classifier was implemented to summarize the multi-omics information and discriminate responders and non-responders. Combining all available radiomic and metabolomic features, the classifier delivered an AUC of 0.864 (± 0.083, p-value < 10–3) with a best-point sensitivity of 90.9% and a specificity of 81.8%. Our results suggest that a multi-omics approach, integrating radiomics and metabolomic data, can enhance the predictive value of standard MRI and could help to avoid unnecessary surgical treatments and their associated long-term complications.

Abstract 5060: Role of molecular driver mutations in recurrence of early stage laryngeal cancer following narrow field definitive radiotherapy

Abstract PURPOSE/OBJECTIVES Recurrent early-stage laryngeal cancer poses profound clinical and prognostic dilemmas. The primary endpoint of this study was to identify patient exome differences between early-stage laryngeal cancer patients who responded or rapidly progressed following definitive, narrow field radiotherapy. Our study offers the first exploration of mutation patterns in early-stage laryngeal head and neck squamous cell carcinoma (HNSCC) patients receiving curative radiation therapy. MATERIALS/METHODS We identified 36 early-stage laryngeal (T1 = 8, T1a = 4, T1b = 2, T1c = 1, T2 = 20, NA =1) HNSCC patients from the University of Kansas Medical Center treated with definitive narrow field radiotherapy to 63-70 Gray (gy) dose. Whole exome sequencing was performed in all patients. Patients were stratified into “responders” (never failed, N=14) and “non-responders” (recurrence &amp;lt;12 months, N=21). Most non-responder patients with local recurrence underwent total salvage laryngectomy (TSL, N=18). All patients underwent whole exome sequencing. The Genome Analysis Toolkit (GATK) was used for pre-processing and functional annotation of variants. Cancer driver genes were detected for each group using the OncodriveCLUST algorithm. Differentially mutated genes were assessed using Fisher’s exact or exact McNemar’s test at P &amp;lt; 0.05. Tumor mutational burden (TMB) comparisons employed the Wilcoxon rank-sum test with the same threshold. For pathway analysis in Reactome and Ingenuity, a P &amp;lt; 0.1 was used for exploratory significance. RESULTS Mutation analysis identified distinct cancer driver genes between responders, (N=3), non-responders (N=36), and post-TSL (N=4). Among 75 genes with significant pre-treatment mutation differences between responders and non-responders, KCNT2 and AGAP6 were exclusively mutated in non-responders (OR=0, P=0.005 and OR=0, P=0.027, respectively), ADAMTS7 was solely mutated in responders (OR=inf, P=0.019), and PLEC showed more mutations in responders (OR=11.6, P=0.006). Pathway analysis showed these genes were involved in the RND2 GTPase cycle, protein O-glycosylation related diseases, and apoptotic pathways. Post-treatment analysis indicated an enrichment of mutations in apoptosis regulation pathways, with genes like PLEC being more frequently mutated in responders than in non-responders or TSL patients. In addition, we observed in non-responders that they trended towards higher median pre-treatment TMB compared to responders (7.98 vs 6.16, P=0.072). There was no significant difference in TMB between TSL patients and responders or non-responders pre-treatment. CONCLUSION Our analysis reveals distinct mutation patterns in early-stage laryngeal cancer patients by response to radiotherapy and the potential role of these mutated genes in apoptosis related pathways. Citation Format: Aaron Segura, Emily Nissen, Chris Lominska, Ryan Morse, Deri Morgan, Prakash Neupane, Dong Pei, Devin Koestler, Randall Kimple, Umamaheswar Duvvuri, Kevin Sykes, Sufi Thomas, Rohit Nallani, Lisa Shnayder, Kiran Kakarala, Emrullah Yilmaz, Andres Bur. Role of molecular driver mutations in recurrence of early stage laryngeal cancer following narrow field definitive radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5060.

Abstract 6687: Evaluation of a new co-cultured microbiome ecosystem therapy candidate (MaaT034) for clinical testing as adjuvant/neoadjuvant to immune checkpoint inhibitors in solid tumors

Abstract Background: Increasing evidence suggests that gut microbiome composition modulates tumor response to therapies, including immune checkpoint inhibitors (ICI). Clinical proofs of concept were obtained in pilot studies using healthy or ICI-responder donor-derived fecal matter transplants to modulate the gut microbiome of non-responding cancer patients and improve their response to ICI. These results support the development of Microbiome Ecosystem TherapiesTM (MET) replicating at large industrial scale the effects of ICI-responders gut microbiome. MaaT Pharma, a clinical-stage biotech, has developed a unique, ground-breaking, patented co-culture process (MET-C). MaaT034, the first-in-class co-cultured microbiome product, has been selected for optimizing intestinal homeostasis and to activate immune functions alone and in combination with an ICI treatment. Method: We combined metagenomic analysis, metabolite quantification by LC-MRM/MS, Caco-2/THP-1 leaky gut model, PBMC assay and mixed lymphocyte reaction (MLR) to assess the impact of MaaT034 on gut homeostasis and immune activation. Results: Metagenomic analysis demonstrates that MaaT034 replicates the richness and diversity of native-based microbiome ecosystems. Metabolite quantification also revealed that MaaT034 microbes secrete anti-inflammatory metabolites such as short chain fatty acids and indoles, as well as metabolites associated with ICI response such as secondary biliary acids. Using a Caco-2/THP-1 leaky gut model, we then demonstrate that MaaT034 conditioned medium was able to restore the integrity of a THP-1-induced damaged gut barrier. This was associated with and a reduction of inflammatory signals. We further performed a human PBMC assay to evaluate the impact of MaaT034 on immune cell activation. We observed the secretion of immunostimulatory cytokines associated with myeloid cell activation in response to MaaT034 conditioned medium. Notably, anti-CD3 mediated T-cells activation was further increased in response to MaaT034 conditioned medium as evidenced by increased IFNγ release associated with increased Granzyme B and CD25. Finally, MLR was performed to determine the immunomodulatory potential of MaaT034 through the interaction between T-cells and monocytes-derived dendritic cells (moDC). Interestingly, MaaT034 conditioned medium improved moDC maturation as well as moDC-induced T-cell activation. MaaT034 conditioned medium further increased anti-PD-1 mediated T-cell activation, as evidenced by IFNγ release. Conclusion: Altogether, these results highlight the potential of MaaT034 to produce metabolites involved in the restoration of gut barrier integrity and stimulation of immune cell response to ICI therapy, with potential benefit for solid tumors treatment. Citation Format: Bastien Laperrousaz, Julie Reygner, Cyrielle Gasc, Charlotte Petitjean, Aurore Duquenoy, Lora Martin, Vincent Bonnaud, Marianne Robin, Sophie Declomesnil, Carole Schwintner, Nathalie Corvaïa. Evaluation of a new co-cultured microbiome ecosystem therapy candidate (MaaT034) for clinical testing as adjuvant/neoadjuvant to immune checkpoint inhibitors in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6687.

Abstract 2876: Spatial immune cell atlas predicts the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable esophageal squamous cell carcinoma

Abstract Combination of radiotherapy and immunotherapy in neoadjuvant treatment seems a promising approach in resectable esophageal squamous cell squamous (ESCC). The clinical study of radiotherapy combined with PD-1 blockade therapy for neoadjuvant treatment of resectable ESCC shows good result. In order to investigate the underlying mechanism of the effectiveness of radiotherapy combined with immunotherapy in ESCC, we collected the pre and post neoadjuvant therapy samples from 5 responders and 5 non-responders ESCC patients and performed multiplexed tissue imaging using 28 markers. Using the detection measurements exported from QuPath, we performed the unsupervised clustering with Seurat and defined 20 cell types. Scimap was used to calculate the distance between different cell types. We found that CD4+ T, CD8+ T, cytotoxic CD8+ T cells and M1 macrophages showed an increase after treatment in both responders and non-responders. Tregs were decreased after neoadjuvant treatment in responder while there was no significant decrease in non-responder. And the distance between Tregs and cancer cells were increased in responders. Collectively, we provide an approach for investigating the underlying mechanism of the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable ESCC using spatial multiplexed imaging. Citation Format: Xiaoyang Yin, Kailun Xu, Shu Zheng, Baosheng Li. Spatial immune cell atlas predicts the sensitivity to neoadjuvant PD-1 blockade treatment combined with radiotherapy in resectable esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2876.

Abstract 3619: Post CAR-T lymphocytosis as a surrogate of BCMA CAR-T expansion, response, and prognosis in multiple myeloma

Abstract Introduction: Expansion of chimeric receptor antibody (CAR)-T cells after infusion has been described as important marker in multiple myeloma (MM); however, the phenomenon of overt lymphocytosis has not been reported. We describe the kinetics of absolute lymphocyte count (ALC) after BCMA CAR-T in MM as a marker of depth of response and progression-free survival (PFS). Methods: 158 patients (pts) were included (93 pts ciltacabtagene autoleucel (cilta-cel) and 65 pts idecabtagene vicleucel (ide-cel) from three institutions. Baseline characteristics, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), ALC on day -5 through 14, and outcomes were collected. A cohort of 84 pts who received CD19 CAR-T for non-Hodgkin lymphoma (NHL) as comparator. Peripheral blood (PB) flow cytometry was performed on days 7, 14 and 21 in 4 patients. Results: Early ALC increase after BCMA CAR-T was frequent, with a median max ALC 1.3 × 103/uL (IQR 1-4.4) from a median baseline of 0. However, when compared to ide-cel, cilta-cel had a higher median Max ALC (2 × 103/uL (1 - 4.4) vs 0.8 (0.7 - 2.8), p &amp;lt; 0.001), longer time-to-max ALC (11 vs 12 days, p = 0.003), and higher absolute ALC increase (2.1 × 103/uL vs 0.7 × 103/uL, p &amp;lt;0.001), while there were no differences in ALC before lymphodepleting conditioning or day 0. Higher max ALC was associated with CRS (OR 4.8, CI 1.3-17.2, p 0.01). Max ALC and absolute increase in ALC were significantly associated with deeper response (VGPR vs PR or less), improved PFS and duration of response (DoR) in univariable analysis. Max ALC &amp;gt;1 × 103/uL (HR 0.4, 0.2-0.7, p &amp;lt;0.001) and non-paraskeletal EMD (HR 2.1, 1.2-.4, p &amp;lt;0.01) remained associated with PFS/DoR in multivariable model after adjusting for CAR-T Product, high-risk cytogenetics, and number of previous lines. PB flow cytometry in 3 responder patients (max ALC range 2-14 × 103) showed expansion of CD3+ BCMA CAR-T from day 7 (BCMA CAR-T &amp;lt;0.2% of CD3+ cells) to day 14 (BCMA CAR-T range 54-88.6%), while a non-responder (max ALC 0.4 × 103) patient had no expansion despite presence of CD3+ BCMA CAR-T at day 7, 14 and 21. BCMA CAR-T had higher max ALC when compared to CD19 CAR-T (median 1.3 × 103 vs 0.4 × 103 (0.2-0.7), p &amp;lt; 0.001), while there were no differences in baseline ALC between BCMA and CD19 CAR-T. Median max ALC did not differ between response status after CD19 CAR-T (0.4 × 103 (0.2-0.7) vs 0.6 (0.3-0.7), p = 0.3). Discussion: Post-CAR-T lymphocytosis is common after BCMA CAR-T therapy for MM and is associated with response, PFS, and DoR after adjusting for other prognostic variables. A cut-off of max ALC &amp;gt; 1 × 103 identified patients with superior PFS, while max ALC &amp;lt;0.5 × 103 represents high-rates of poor response and early progression. ALC expansion correlated with BCMA CAR-T expansion as assessed by flowcytometry, supporting ALC assessment as an accessible surrogate that is clinically applicable and accessible. Citation Format: Mateo Mejia Saldarriaga, Darren Pan, Caitlin Unkenholz, Tarek Mouhieddine, Josh Fein, Jorge Monge, Cara Rosenbaum, Roger Pearse, David Jayabalan, Christian Gordillo, Hei Ton Chan, Markus Mapara, Giorgio Inghirami, Suzanne Lentzsch, Ren Reshef, Adriana Rossi, Samir Parekh, Sundar Jagannath, Shambavi Richards, Ruben Niesvizky, Mark Bustoros. Post CAR-T lymphocytosis as a surrogate of BCMA CAR-T expansion, response, and prognosis in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3619.

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4559.

Abstract 1375: Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy

Abstract Background: Intra-tumoral CD4+ and CD8+ T cells expressing inhibitory receptor CD161 have an effector memory phenotype with high cytotoxic potential. CD161 inhibits T and NK cell function in the presence of its ligand, CLEC2D. This suppression can be recovered by treatment with IMT-009, a first in class anti-CD161 antibody. Single cell transcriptomic analysis of anti-PD1 refractory microsatellite stable colorectal tumors identified presence of CD161+ CD4+ cytotoxic T cells and CD161+ PD1+ exhausted CD8 T cells suggesting that anti-CD161 treatment may provide benefit in these tumors both as a monotherapy and in combination with anti-PD1. It was also observed that CD161+ CD4/CD8T cells are more abundant in anti-PD1/L1 non responders compared with responders in hepatocellular carcinoma, cutaneous squamous cell carcinoma, clear cell renal carcinoma and breast cancer, providing a rationale for targeting CD161. Using engineered ex-vivo tumor models and dissociated human tumors, we have demonstrated that IMT-009 reverses CLEC2D mediated inhibition and restores primary T cell function. Additionally, co-treatment of IMT-009 with anti-PD1 further enhanced T cell mediated cytotoxicity, providing rationale for studies combining IMT-009 and anti PD1 in the clinic. Methods: Using the Snakemake pipeline developed in house, we processed publicly available scRNAseq data. Cells were clustered by Louvian method followed by cell type annotation by Seurat, and gene expression and gene signature levels per cluster were plotted. For ex-vivo assays, antigen specific CD8 T cells expanded from HLA-A2:01 donors were co-cultured with HLA-A2:01 expressing MDA-MB 231 triple negative breast cancer cell line or Raji Burkitt Lymphoma model with HLA-A2:01 allelic overexpression. Target tumor cells were engineered to express the ligands CLEC2D or PDL1. These cell lines were made to overexpress luciferase for monitoring target cell death using a luminescence readout. Additionally, dissociated tumor cells (DTCs) were cultured in presence of the treatment molecules. Intracellular staining of cytotoxic T cells along with detection of IFNγ, IL-2, TNFα and Granzyme B levels in culture supernatants by MSD/ELISA were used to assess activation profile of tumor infiltrated T cells. Results: Combining anti-PD1 and IMT-009 significantly enhanced cytotoxic benefit compared to individual monotherapy in both Raji and MDA-MB-231 target cell line models. IFNγ levels in supernatants of DTCs treated with this combination were elevated as compared to individual monotherapy. Together with scRNAseq data of increased abundance of KLRB1 (CD161) expressing T cells in anti PD1 non-responder patients, our data provide rationale for testing combination of IMT-009 with anti-PD1 in patients that may not respond to anti PD1 alone. Citation Format: Adwitiya Kar, Tim Nieuwenhuis, Meixue Duan, Siamak Tabrizi, Matthew Huggins, Elizabeth Scanlon, Cynthia Hession, Matthew Bernstein, Michael Ross, Ming Tang, Shruti Malu. Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1375.

Abstract 4170: Artificial intelligence (AI)-based multi-modal approach using H&amp;E and CT image for predicting treatment response of immune checkpoint inhibitor (ICI) in non-small cell lung cancer (NSCLC)

Abstract Background: Previously, AI-powered spatial tumor-infiltrating lymphocyte analysis in H&amp;E whole-slide image (WSI) showed promising performance in predicting ICI outcomes. However, there were several innate limitations of tissue analysis, including the time lag between tissue biopsy and actual ICI administration, during which tumor microenvironment (TME) has changed, and analyzing a part of a single tumor tissue would not fully assess immunogenicity of multiple metastatic tumors. To overcome the challenges, we developed multiple-instance learning (MIL)-based models and applied them to predict the treatment response of ICI in a real-world dataset. Method: MIL-based models were trained under the cross-validation scheme for CT scans (N=456) separately, based on CT image analyzers. Lunit SCOPE IO (Lunit, South Korea), an AI-based H&amp;E WSI analyzer, identifies various classes of cells within TME and segments tumor regions from WSI (AI-HE-TIL). An AI-based CT image analyzer detects nodules on the CT image (AI-CT). The final ensemble score per patient is computed by the average of all model predictions from CT and pathology image inputs. Single and ensemble performances for predicting responder and non-responder patients to ICI are measured in an independent cohort of 208 NSCLC patients. Results: In the overall cohort, the AUROC values for AI-HE-TIL, AI-CT, and the ensemble were 0.6372, 0.6759, and 0.7307, respectively. The high ensemble score group (≥ median) has prolonged progression-free survival (PFS) of ICI (median PFS 5.0 months vs 2.1 months, hazard ratio [HR] 0.54, P &amp;lt; 0.0001). It correlates with PD-L1 expression, as tumor proportion score (TPS) ≥ 50% subgroup had higher ensemble score (median 0.312 vs 0.257, P = 0.0295). Interestingly, among TPS ≥ 50% subgroup, the high ensemble score shows significantly favorable PFS of ICI (median PFS 6.7 months vs 2.9 months, HR 0.46, P = 0.0002). In subgroup analysis according to tissue harvest site (primary tumor vs lymph node or distant metastasis), the AUROC values of AI-HE-TIL and AI-CT for the subgroup harvested from the primary tumor (N=111) were 0.6568 and 0.6658, respectively, and those for the subgroup harvested from the metastatic lesion (N=97) were 0.6162 and 0.6973, respectively, implying AI-CT is relatively not affected by tissue harvest site. Conclusion: AI-powered multi-modal approach of using H&amp;E image and CT image shows complimentary, and synergistic effect to predict ICI clinical outcomes in advanced NSCLC, even in the subgroup of PD-L1 high population. Citation Format: Dong Young Jeong, Jongchan Park, Heon Song, Jimin Moon, Taebum Lee, Changho Ahn, Sehhoon Park, Se-Hoon Lee, Chan-Young Ock, Ho Yun Lee. Artificial intelligence (AI)-based multi-modal approach using H&amp;E and CT image for predicting treatment response of immune checkpoint inhibitor (ICI) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4170.

Abstract 68: Imaging Endothelial PD-L1 expression with ultrasound molecular imaging to stratify immune checkpoint inhibitor responders

Abstract Immune checkpoint blockade therapies (ICTs) have revolutionized oncology through substantial and lasting treatment outcomes, but only in 15-20% of patients. Non-invasive diagnostic tools to select patients that are most likely to respond to ICT and guide treatment regimens, and thus minimize toxicities to non-responder patients, are in high demand. Program death-1 ligand (PD-L1) marker evaluation in the tumor vasculature and tumor microenvironment is an essential criterion for identifying a suitable patient for PD-L1 based ICT, and it also serves as a prognostic marker in tumor response evaluation. Ultrasound molecular imaging (USMI) is an up-and-coming non-invasive imaging modality that enables longitudinal characterization of endothelial-specific markers. We are developing USMI methods to image immune markers expressed specifically on endothelial cells (EC) of tumors as a decision-support tool for guiding ICT strategies. In this work, we demonstrate for the first time the potential of imaging EC-specific immunosuppressive markers such as PD-L1 and investigate whether it can act as a marker for predicting ICT response. Target-ready microbubble (MB) was used (Trust Bio-sonics, Taiwan) to generate PD-L1 antibody (Ab) linked MBs (TMBs). DSPE-PEG was functionalized to anti-PD-L1 Ab or isotype-Ab (control) to generate stable TMBs (~5 × 107 MBs/mL) to target PD-L1 expressing ECs in vivo. We studied the treatment effect of anti-PD-L1-Ab and isotype-Ab using a syngeneic CT-26 tumor in mice and demonstrated the targeting potency of TMB or iso-Ab-MB (NMB). US imaging was performed using Vevo 2100 high-frequency US system with M250 transducer to evaluate and quantify the differentially targeted enhancement parameter (dTE). We observed that TMBs bound PD-L1 expressing ECs in vivo, with high and low enhancement for TMB and NMB, respectively, in tumors validated by dTE quantification through characterization of the slower washout kinetics. Furthermore, TMBs exhibited increased tumor retention and prolonged binding with average differential targeted enhancement values (dTE) (+/- SD) of 5.2 ± 7.4 for TMBs compared to 2.4 ± 3.9 for NMBs (p=0.14). Preliminary results of USMI (dTE values of TMBs) indicate that PD-L1-treated mice showed a higher response (4.5 ± 5.2; n=9) to the treatment group than control mice that did not receive Ab (1.4 ± 0.8; n=4). The tumor growth rate for the treated mice group was 5.4 -fold retarded compared to the control group which correlated with USMI dTE values. The USMI signals correlated with IHC performed on tumor tissue ex vivo from the respective mouse used for US imaging. Overall, our pre-clinical study suggests that USMI using TMBs may contribute to non-invasive assessment of IC expression on tumor ECs, and that it may serve as a predictive biomarker of ICT response. This novel TMB agent could be a game changer for CE USMI-based cancer prognosis in ICB therapy. Citation Format: Arutselvan Natarajan, Farbod Tabesh, Mahsa Bataghva, Ramasamy Paulmurugan, Ahmed Nagy El Kaffas. Imaging Endothelial PD-L1 expression with ultrasound molecular imaging to stratify immune checkpoint inhibitor responders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 68.

Abstract 6686: The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors

Abstract Gut microbiota is associated with antitumor efficacy of immune checkpoint blockade therapies, yet the mechanism is not largely understood. Here, we show that a novel bacterial strain of Ruminococcaceae isolated from the feces of patients who respond to PD-1 (programmed cell death 1) blockade efficiently primes/activates tumor-specific CD8+ T cells through effective stimulation/maturation of CD103+ dendritic cells (DCs) during migration from the gut to the tumor microenvironment (TME). The administration of this bacteria strain with PD-1 blockade significantly inhibited tumor growth, even when adding them to fecal transplantation of non-responder patients in animal models. Mechanistically, the bacterial strain promoted the IRF8-driven differentiation of CD103+ DCs by stimulating multiple Toll-like receptors and the accumulation of these DCs in tumors and tumor-draining lymph nodes. These CD103+ DCs prolonged engagement with cognate antigen-specific CD8+ T cells, which induced the activation of CD8+ T cells specific for a wide variety of tumor antigens and fostered their PD-1 expression through enhanced T-cell activation signals with NFATc1 nuclear translocation, thereby leading to abundant PD-1+CD8+ T cells, a key effector cell population in PD-1 blockade, with a broader T-cell receptor repertoire in the TME. Our findings with novel single bacterium isolated from feces of responder patients demonstrate the mode of action by gut microbiota to augment antitumor immunity. Citation Format: Nina Yi-Tzu Lin, Shota Fukuoka, Daisuke Motooka, Yuko Shigeno, Takuma Irie, Dieter M. Tourlousse, Kazutoshi Murotomi, Yuji Sekiguchi, Hideyuki Tamaki, Mariko Shindo, Takahiro Tsuji, Hiroaki Wake, Koichi Goto, Toshihiko Doi, Kohei Shitara, Keisuke Watanabe, Yuka Maeda, Shota Nakamura, Yoshimi Benno, Hiroyoshi Nishikawa, Shohei Koyama. The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6686.

Abstract 1268: Immune activation characterization via amino acid concentration signatures for multi-cancer early detection and CDKi treatment response prediction

Abstract Early detection of cancer can increase survival rates by 5-10 times. In the US, a significant portion of cancers are still detected at a late stage, for example, 32.1% of breast cancer, 45.5% of lung cancer, 22.8% of colon cancer patients were diagnosed at advanced stage. Early detection is challenging because many cancers are asymptomatic in early stages, although cancer screening can detect cancer before symptoms emerge. Current standard-of-care screening programs are limited and directed to single types of cancer. Non-invasive multi-cancer early detection (MCED) is urgently needed because it can screen for several cancer types with a single blood test, increasing cancer screening coverage while avoiding cumulative false positive rates. Developmental MCED approaches usually rely on detection of circulating tumor DNA (ctDNA) released from tumors. However, ctDNA signals scale with tumor burden limiting sensitivity and specificity, particularly for early-stage cancers, while also limiting applicability to precision oncology. Here we report a MCED approach with superior sensitivity and specificity, which can also be used to predict treatment response. We introduce a novel strategy for detecting cancers from blood plasma, profiling the immune response to disease development rather than relying on detection of emerging diseased tissue. The platform is designed to detect alterations in the ratio of immunoglobulins and albumin, as well as class-switching among immunoglobulins, by measuring the overall concentration of amino acid residues incorporated into proteins in patient plasma. By using our biorthogonal chemistry labeling tool, we were able to measure and characterize amino acid concentration signatures (AACS) within the neat blood samples, including cysteine, free cysteine, lysine, tryptophan and tyrosine, which allowed us to observe distinct signatures for varying cancers including breast, prostate, colorectal and pancreatic cancers. We could identify 84% of cancers with &amp;lt;0.5% false positive rate (N=93). In this study, cancer AACS signatures were distinct from signatures measured with non-cancerous immune activation, including autoimmune diseases and infection. Moreover, AACS was also correlated to clinical features, such as cancer metastatic statuses and therapy response. With Cyclin-dependent Kinase 4/6 Inhibitors-treated advanced breast cancer patients as an example, we achieved 100% correct prediction of responding patients and 91.7% accurate predictions of non-responding patients by using AACS in treatment-naïve samples. Altogether, the novel AACS approach is a powerful combination of bioorthogonal chemistry and machine learning analysis in clinical practice, and it can be potentially used for cancer screening and support clinical decisions for treatment selection and patient stratification in new indications. Citation Format: Cong Tang, Patrícia Corredeira, Sandra Casimiro, Wesley Sukdao, Luís Costa, Emma Yates, Gonçalo Bernardes. Immune activation characterization via amino acid concentration signatures for multi-cancer early detection and CDKi treatment response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1268.