Biomarkers Of Clinical Response Research Articles

Cerebrospinal Fluid Biomarkers Associated with Intrathecal Cell Therapy in Patients with Progressive Multiple Sclerosis (P1-3.001)

<h3>Objective:</h3> To identify cerebrospinal fluid (CSF) biomarkers of clinical response to intrathecal (IT) mesenchymal stem cell-neural progenitor (MSC-NP) treatment in patients with progressive multiple sclerosis (MS). <h3>Background:</h3> MSC-NPs are a bone marrow-derived population of cells with trophic and immunomodulatory properties with therapeutic potential in MS. A phase II randomized, double-blind, placebo-controlled clinical trial was performed to investigate whether multiple intrathecal (IT) injections of autologous MSC-NPs was associated with clinical efficacy. Study subjects received either 6 IT injections of autologous MSC-NPs or 6 placebo injections spaced 2 months apart. In the crossover study design, subjects treated with placebo in the first year were treated with MSC-NPs in the second year, and vice versa. Safety and clinical efficacy will be reported elsewhere. <h3>Design/Methods:</h3> For biomarker discovery, CSF was collected from 36 study subjects at the time of their first and sixth IT-MSC-NP treatment reflecting pre-treatment and post-treatment timepoints, respectively. CSF supernatants were analyzed using SOMAScan multiplex proteomic assay. Differentially expressed proteins were identified using a multivariate linear mixed effect model. Biomarkers were validated using ELISA/Luminex assays. Treatment responders were defined by improvement in any of the following outcomes: EDSS, timed 25-foot walk, 9-hole peg test, 6-minute walk test, or muscle strength. <h3>Results:</h3> Proteomic analysis of CSF identified matrix metalloproteinase-9 (MMP-9), complement C3a and the chemokine CCL2 as potential biomarkers of IT-MSC-NP treatment. MMP-9 was significantly increased post-treatment and was observed independent of clinical response. Relative concentration of C3a, C3 and C3b were decreased post-treatment and subgroup analysis demonstrated significance only in the responder group. CCL2 was significantly decreased post-treatment with higher significance associated with clinical response. Decreased CCL2 was validated in an independent cohort of patients treated with IT-MSC-NPs. <h3>Conclusions:</h3> These results identify multiple CSF biomarkers of IT-MSC-NP treatment that may correlate with therapeutic response to this novel cell therapy in MS. <b>Disclosure:</b> Miss Kizilbash has nothing to disclose. Ms. Carlson has nothing to disclose. Violaine K. Harris, PhD has nothing to disclose. Dr. Sadiq has nothing to disclose.

Fronto-central resting-state 15-29 Hz transient beta events change with therapeutic transcranial magnetic stimulation for posttraumatic stress disorder and major depressive disorder

Repetitive transcranial magnetic stimulation (rTMS) is an established treatment for major depressive disorder (MDD) and shows promise for posttraumatic stress disorder (PTSD), yet effectiveness varies. Electroencephalography (EEG) can identify rTMS-associated brain changes. EEG oscillations are often examined using averaging approaches that mask finer time-scale dynamics. Recent advances show some brain oscillations emerge as transient increases in power, a phenomenon termed “Spectral Events,” and that event characteristics correspond with cognitive functions. We applied Spectral Event analyses to identify potential EEG biomarkers of effective rTMS treatment. Resting 8-electrode EEG was collected from 23 patients with MDD and PTSD before and after 5 Hz rTMS targeting the left dorsolateral prefrontal cortex. Using an open-source toolbox (https://github.com/jonescompneurolab/SpectralEvents), we quantified event features and tested for treatment associated changes. Spectral Events in delta/theta (1–6 Hz), alpha (7–14 Hz), and beta (15–29 Hz) bands occurred in all patients. rTMS-induced improvement in comorbid MDD PTSD were associated with pre- to post-treatment changes in fronto-central electrode beta event features, including frontal beta event frequency spans and durations, and central beta event maxima power. Furthermore, frontal pre-treatment beta event duration correlated negatively with MDD symptom improvement. Beta events may provide new biomarkers of clinical response and advance the understanding of rTMS.

Abstract 5112: Immune biomarkers of clinical response after NSAID treatment in combination with vaccination targeting EGFR-COX2-CDC25B in the APC(Min/+) mouse model

Abstract Studies from our group have shown that administration of NSAIDs in mouse models of Familial Adenomatous Polyposis (FAP) results in a decrease in the development in small intestinal polyps with a significant increase in CD8 tumor infiltrating lymphocytes (TIL) and downregulation of PD-L1 on tumors. Furthermore, we have demonstrated that NSAIDs could be used in combination with vaccination to even further reduce the development of intestinal polyps. We questioned what tissue specific biomarkers could define the efficacy of response with the combination treatment. APC(Min/+) male and female mice (4-6 weeks old) were immunized with a multi-antigen (EGFR, COX2, CDC25B) peptide vaccine with CFA/IFA as an adjuvant. Control mice received immune adjuvant alone. Regular chow or chow containing 400 ppm naproxen was provided intermittently (3 weeks on, 3 weeks off for 3 cycles) or continuously (18 weeks on) at the initiation of the vaccine regimen. Animals were euthanized at 22 weeks and tumors quantified. TIL were evaluated by flow cytometry. Since intermittent naproxen treatment was just as effective at inhibiting tumor development as continuous naproxen treatment, with or without vaccine, we did not differentiate between NSAID regimens. We observed an increase in CD8+ (83%; p&amp;lt;0.0001), Tbet+CD4+ (94%; p&amp;lt;0.001), effector memory (EM) CD4+ (76%; p&amp;lt;0.0001) and EM CD8+ (94%; p&amp;lt;0.0001) TIL in the tumors that did develop in the mice treated with the combination NSAID and vaccination as compared to control. There was increased CD8+ (Pearson correlation coefficient: −0.506, p&amp;lt;0001), Tbet+CD4+ (Pearson correlation coefficient: -0.6222, p&amp;lt;0.001), EM CD4+ (Pearson correlation coefficient: 0.-6074, p&amp;lt;0.001) and EM CD8+ (Pearson correlation coefficient: -0.7155, p&amp;lt;0.001) in mice with greater tumor inhibition than those with less protection. Receiver operator curves revealed that CD8+ (AUC: 0.7623; p=0.002), Tbet+CD4+ (AUC 0.7273; p=0.008), EM CD4+ (AUC 0.6782; p=0.04) and EM CD8 (AUC 0.7897; p&amp;lt;0.001) TIL can differentiate mice receiving the combination of NSAID and vaccination as compared to either monotherapy alone. These data define potential biomarkers of response that can guide human clinical trials of NSAIDs +/- vaccination in patients with FAP. Citation Format: Denise L. Cecil, Ying Liu, Lauren Corulli, Mary L. Disis. Immune biomarkers of clinical response after NSAID treatment in combination with vaccination targeting EGFR-COX2-CDC25B in the APC(Min/+) mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5112.

Abstract 2130: Lipid and immune-based biomarkers associated with clinical response to TPST-1120: A small molecule antagonist of peroxisome-proliferator activated receptor-alpha

Abstract Background: TPST-1120 is a small molecule antagonist of peroxisome-proliferator activated receptor-alpha (PPAR-α), a regulator of fatty acid oxidation and immune suppression. TPST-1120 was well tolerated and showed signs of activity in a phase I trial as monotherapy and in combination with nivolumab (NCT03829436). The objective response rate was 30% (3/10, all partial responses) in subjects treated at the two highest TPST-1120 doses in combination with nivolumab and included two subjects with renal cell carcinoma previously refractory to anti-PD-1 therapy [1]. We performed ctDNA mutational analysis at baseline and quantified lipid and gene expression changes in post-treatment whole blood to identify potential biomarkers of response. Methods: Mutational analysis of ctDNA was assessed using the PredicineCARE™ assay (Predicine Inc.), and lipid analysis was performed by tandem mass spectrometry. Gene expression changes were quantified using the nCounter® PanCancer Immune Profiling panel (NanoString Inc.) supplemented with 30 PPAR-α target genes. Putative clinical response biomarkers were identified as those differentially expressed by patients with partial response (PR) compared to those with progressive disease (p&amp;lt;0.05 by Mann-Whitney U Test). Longitudinal lipid change magnitudes were assessed by Wilcoxon paired analysis (p&amp;lt;0.05). Results: Baseline ctDNA mutational analysis revealed that patients with PR or stable disease were more likely to bear mutations in isocitrate dehydrogenase (IDH) and phosphatase and tensin homolog (PTEN) compared to patients with progressive disease. Patients with PR demonstrated significant elevations (p&amp;lt;0.05) in multiple genes including those associated with lipid transport (APOE), Th17 development (RORC) and down-regulation of CD155, a TIGIT ligand. Lipid analysis demonstrated acute changes in free fatty acids (FFA) four hours after first dose (p&amp;lt;0.05). Among patients receiving combination therapy, the highest post-dose elevations in FFA, lysophosphatidylcholine and lysophosphatidylethanolamine levels were observed in a PR patient exhibiting the longest duration of response. Conclusions: TPST-1120 treated patients with PR demonstrated fatty acid oxidation perturbations and immune gene expression changes as potential biomarkers of clinical benefit. Increased frequencies of responding patients bearing PI3K pathway or IDH mutations may reveal populations likely to benefit from treatment with TPST-1120. 1. Yarchoan M, et al., “A phase 1 study of TPST-1120 as a single agent and in combination with nivolumab in patients with advanced solid tumors.” Journ. Clin. Onc. 2022; 40 (16) suppl. Citation Format: Nathan E. Standifer, Yonchu Jenkins, Sam Whiting, Thomas W. Dubensky. Lipid and immune-based biomarkers associated with clinical response to TPST-1120: A small molecule antagonist of peroxisome-proliferator activated receptor-alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2130.

Immunologic Predictors for Clinical Responses during Immune Checkpoint Blockade in Patients with Myelodysplastic Syndromes.

The aim of this study is to determine immune-related biomarkers to predict effective antitumor immunity in myelodysplastic syndrome (MDS) during immunotherapy (IMT, αCTLA-4, and/or αPD-1 antibodies) and/or hypomethylating agent (HMA). Peripheral blood samples from 55 patients with MDS were assessed for immune subsets, T-cell receptor (TCR) repertoire, mutations in 295 acute myeloid leukemia (AML)/MDS-related genes, and immune-related gene expression profiling before and after the first treatment. Clinical responders treated with IMT ± HMA but not HMA alone showed a significant expansion of central memory (CM) CD8+ T cells, diverse TCRβ repertoire pretreatment with increased clonality and emergence of novel clones after the initial treatment, and a higher mutation burden pretreatment with subsequent reduction posttreatment. Autophagy, TGFβ, and Th1 differentiation pathways were the most downregulated in nonresponders after treatment, while upregulated in responders. Finally, CTLA-4 but not PD-1 blockade attributed to favorable changes in immune landscape. Analysis of tumor-immune landscape in MDS during immunotherapy provides clinical response biomarkers.

Non-Coding RNA Investigations in Cutaneous Melanoma: A Step forward in Discovering Novel Biomarkers

Non-Coding RNA Investigations in Cutaneous Melanoma: A Step forward in Discovering Novel Biomarkers

Neural Correlates of the DEEPP (Anti-suicidal Response to Ketamine in Treatment-Resistant Bipolar Depression) Study: Protocol for a Pilot, Open-Label Clinical Trial.

Suicide is among the top 10 leading causes of death worldwide. Of people who died by suicide, the majority are diagnosed with depression. It is estimated that 25%-60% of people with bipolar depression (BD) will attempt suicide at least once, and 10%-15% will die by suicide. Several treatments, such as lithium, clozapine, electroconvulsive therapy, and cognitive behavioral therapy, have been shown to be effective in treating suicidality. However, these treatments can be difficult to tolerate or may take months to take effect. Ketamine, a glutamate N-methyl-D-aspartate antagonist, has been shown to have rapid antisuicidal effect and antidepressant qualities, and is thus a promising intervention to target acute suicidality in patients with BD. However, the biological mechanism underlying its therapeutic action remains poorly understood. Enhancing our understanding of underlying mechanisms of action for ketamine's effectiveness in reducing suicidality is critical to establishing biological markers of treatment response and developing tailored, personalized interventions for patients with BD. This is an open-label clinical trial to test the safety and feasibility of repeated ketamine infusions to treat acute suicidality. The primary objective is to test the safety and feasibility of ketamine intervention. The secondary objective is to examine ketamine's potential neurophysiological mechanisms of action by assessing cortical excitation and inhibition to determine potential biomarkers of clinical response. Other objectives are to evaluate the effect of ketamine on acute suicidality and other clinical outcomes, such as depressive symptoms and quality of life, to inform a future larger trial. This open-label clinical trial aims to test the safety and feasibility of repeated ketamine infusions in patients with BD for suicidality and to assess ketamine's neurophysiological effects. A sterile form of racemic ketamine hydrochloride will be administered over a 40-minute intravenous infusion 2 times per week on nonconsecutive days for 4 weeks (8 sessions). We will recruit 30 adults (24-65 year olds) over 2 years from an academic psychiatric hospital in Toronto, Canada. This study is currently ongoing and actively recruiting participants. So far, 5 participants have completed the trial, 1 is currently in active treatment, and 8 participants are on the waitlist to be screened. We anticipate initial results being available in the fall of 2023. This proposal was presented as a poster presentation at the Research to Reality Global Summit on Psychedelic-Assisted Therapies and Medicine, held in May 2022 in Toronto, Canada. Developing effective interventions for acute suicidality in high-risk populations such as those with BD remains a major therapeutic challenge. Ketamine is a promising treatment due to its rapid antidepressant and antisuicidal effects, but its underlying neurophysiological mechanisms of action remain unknown. ClinicalTrials.gov NCT05177146; https://clinicaltrials.gov/ct2/show/NCT05177146. DERR1-10.2196/41013.

The negative effect of antibiotics on RCC patients with immunotherapy: A systematic review and meta-analysis.

Microbiome dysbiosis is considered a predictive biomarker of clinical response in renal cell carcinoma (RCC), which can be regulated by antibiotics (ATB). Multiple studies have shown that concomitant ATB administration has inhibitory effects on immunotherapy in RCC. This review aimed to assess the impact of ATB on patient survival and tumor response in RCC with immunotherapy. Literature evaluating the effect of ATB on immunotherapy in RCC from Cochrane Library®, PubMed®, Embase®, Scopus®, and Web of Science® were systematically searched. Hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS), odds ratio (OR) for objective response rate (ORR) and primary progressive disease (PD) were pooled as effect sizes for clinical outcomes. Subgroup analysis was conducted to reveal the determinants of the effect of ATB on immunotherapy, including time windows of ATB exposure to immunotherapy initiation, ICIs treatment and study location. The leave-one-out approach was adopted to analyze the heterogeneity formulated. Cumulative meta-analysis adding by time was used to observe dynamic changes of the results. Ten studies were included in the systematic review and six studies (with n=1,104 patients) were included in the meta-analysis, four studies were excluded for overlapping patients with subsequent larger studies and lack of unique patient-level data. ATB administration was significantly correlated with shorter PFS (HR=2.10, 95%CI [1.54; 2.85], I2 = 2% after omitting study Derosa et al, 2021 detected by leave-one-out approach), shorter OS (HR=1.69, 95%CI [1.34; 2.12], I2 = 25%) and worse ORR (OR=0.58, 95%CI [0.41; 0.84]), but no difference was observed in risk of PD (OR=1.18, 95%CI [0.97; 1.44]). No significant differences existed among the subgroups for determining the determinants of ATB inhibition. Concomitant ATB with immunotherapy was associated with worse PFS, OS and ORR in RCC. No publication bias was observed in this study. https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=349577, identifier CRD42022349577.

115 The frequency of intermediate monocytes before treatment is a candidate biomarker of clinical response to secukinumab in of psoriasis

Biological therapies have transformed psoriasis treatment, however patient response can vary. Secukinumab, which targets interleukin(IL)-17A, is highly effective with 57.4% of psoriasis patients achieving a 90% reduction in psoriasis area and severity index (PASI90) in real world data. Nevertheless, over 40% of patients may respond better to alternative treatments, highlighting a need for predictive biomarkers of response to stratify patients. To this end, we performed deep-immunophenotyping of myeloid cells from peripheral blood mononuclear cells (PBMCs) from patients at baseline and 12 weeks following secukinumab treatment (w12) (n=17).

Spatial Protein Analysis of the Tumor Microenvironment and Biomarkers in Hodgkin's Lymphoma

Introduction: The tumor microenvironment (TME) is the complex milieu of cells and molecules surrounding the tumor. Single cell methods have been used to great effect to identify subpopulations of cells that have pro- or anti-tumor properties, and selectively modulating these has great therapeutic benefits, especially in immunotherapy. However, there is limited information on the spatial organization of these subpopulations, which determines how they signal and their therapeutic potential. Single cell resolved spatial computational analysis is needed to describe the complex interactions of the TME and their effect on patient outcomes. Hodgkin's Lymphoma presents a unique spatial TME due to the sparse tumor distribution of Hodgkin's Reed-Sternberg tumor cells. Yet even though it is difficult to disentangle signaling in Hodgkin's tumor cells - which are immune derived - from the immune TME, Hodgkin's is highly receptive to checkpoint inihibitors among lymphomas and insights gleaned from the Hodgkin's TME could better inform immunotherapies across lymphomas. Materials and Methods: Here we apply Imaging Mass Cytometry (IMC), a technology to perform ~40-plex protein analysis with 1 micron resolution in tissue, to study a cohort of 260 matched samples at diagnosis and after relapse from 90 patients with relapsed/refractory Hodgkin's Lymphoma. We developed a computational pipeline to perform cell phenotyping, spatial analysis, and biostatistics, to describe tumor architecture and propose putative biomarkers of Hodgkin's clinical response and relapse. A novel feature of the pipeline is to quantify proteins and spatial analysis on the same numerical scale for each cell, to generate hybrid biomarkers. Results and Discussion: We analyzed over 7 million cells for their phenotype and spatial organization. We use IMC to describe spatial features of the tumor - cell subtypes and their positioning - that correlate to clinical outcomes. We identify proteins such as CXCR5 that correlate to survival in specific spatial contexts, and we describe spatial reorganization from diagnosis to the relapsed tumor as it relates to survival and relevant clinical factors such as MHC expression and EBV infection. A significant conceptual advance was to use spatial metrics to perform "digital biopsies", a selection of tumor regions comparable across patients. We also describe cell rosetting, a localized recruitment of immune cells to tumor that is characteristic of Hodgkin's. We validated multiple existing biomarkers in the literature using our data set and proposed novel biomarkers using IMC data. Conclusions: Spatial analysis of the HL microenvironment revealed composite features of the TME that predict clinical outcomes. These features cannot be described using single cell tools or low-plexed imaging, and represent a truer picture of HL biology. The pipeline developed here can be universally applied to other spatial protein data for biomarker discovery and analysis, and the biomarkers proposed here will be validated with IHC. Figure caption: IMC analysis of Hodgkin's Lymphoma. A. IMC generates a 40-plex protein image. B, C. The image is converted to single cells and analyzed for protein and spatial features. D. A UMAP of cell phenotypes and the spatial interactions of specific phenotypes. E. The spatial and protein values are used to generate predictive biomarkers of clinical response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD25, Soluble CD25, and CCL17 As Potential Predictors of Clinical Response to Camidanlumab Tesirine in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma

Introduction: Camidanlumab tesirine (Cami), an antibody-drug conjugate targeting CD25, has shown single-agent anti-tumor activity and manageable toxicity in a phase 2 trial in relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL; Carlo-Stella, et al. 2022). In the phase 1 cHL trial, biopsy tumor cell CD25 histoscores (H-scores) were significantly higher in responders vs nonresponders and low baseline soluble CD25 (sCD25) was possibly related to response (Hamadani, et al. 2021). Separately, circulating CCL17 has been correlated with response to therapies for cHL (Plattel, et al. 2016). Objective: To assess CD25, sCD25, and CCL17 as predictive biomarkers of clinical response to Cami in patients (pts) with R/R cHL. Methods: This analysis was based on the open-label, multicenter, phase 2 study in pts with R/R cHL after ≥3 prior lines of therapy (or ≥2 in pts ineligible for SCT; NCT04052997). CD25 expression was assessed by immunohistochemistry (IHC) and H-score in all archival tumor biopsies and a subset of recent biopsies (archival biopsies taken after the last systemic anticancer treatment, including SCT if applicable); serum sCD25 and CCL17 were assessed by qualified immunoassays prior to Cami infusion on day 1 of each cycle. Cami may interfere with the sCD25 assay, underestimating post-baseline (BL) sCD25. CD25 IHC, BL sCD25 and CCL17, and changes in sCD25 and CCL17 from BL were compared between responders and nonresponders. Receiver operating characteristic (ROC) analyses were performed to evaluate the predictive potential of these measures as response biomarkers. Results: Assessments of CD25, sCD25, and CCL17 in pts with a best overall response (BOR) of complete or partial response (responders) and stable disease or PD (nonresponders) were performed for 111 of 117 pts treated with Cami (6 pts were not evaluable for BOR). No significant difference in tumor cell CD25 H-score was observed between responders and nonresponders in all (n=99) or recent biopsies (n=23; p>0.05 for both), despite higher median levels in responders. BL sCD25 (n=108) was significantly higher in nonresponders than responders (p=0.018; Fig 1); though, overlapping values were observed in a substantial number of pts. There was also a significantly greater decrease in sCD25 in nonresponders vs responders from BL to C3D1 (n=79; median 0.53 vs 1.02; p=0.018). While there was no significant difference in BL CCL17 levels (n=106), there was a significantly greater decrease in BL CCL17 in responders vs nonresponders at C2D1 (n=97; median 0.50 vs 0.81; p<0.01 log10 change). In ROC analyses in a subset of 96 pts with CD25 IHC and BL sCD25 results available, sCD25 alone showed a modest predictive potential (area under curve [AUC]=0.65) that was not improved with the addition of tumor cell CD25 H-score (AUC=0.67). In a subset of 76 pts with results available for sCD25 and CCL17 at BL and C2D1, ROC curves of BL sCD25, BL sCD25 plus BL CCL17, sCD25 change from BL, and CCL17 change from BL showed modest predictive potential (AUC<0.7 for all). A better AUC was found in a model including change from BL at C2D1 of sCD25 and CCL17 (AUC=0.74). This AUC was further increased in a model of BL sCD25 and CCL17 plus their change from BL at C2D1 and a model of BL sCD25 plus sCD25 and CCL17 change from BL at C2D1 (AUC=0.76 for both; Fig 2). The latter model had a higher Youden index. Conclusions: Higher median tumor cell CD25 H-score was seen in responders vs nonresponders in all and recent biopsies but the differences did not reach significance. These data warrant further investigations as phase 1 data showed significantly higher tumor CD25 expression in responders (Hamadani, et al. 2021). Nonresponders had significantly higher BL sCD25; however, this parameter alone has insufficient predictive potential for Cami efficacy. Predictive models including BL sCD25 plus sCD25 and CCL17 change from BL at C2D1 (± BL CCL17) showed the best ROC AUCs but need further refining to be clinically relevant. However, given the high overall and early responses in most pts treated with Cami, biomarkers predicting response at C2D1 are suboptimal unless reaching a higher sensitivity and specificity. Investigation of sCD25 in the context of the Cami mode of action and exposure-response and the potential value of sCD25 and CCL17 changes as early indicators of Cami's effect and as biomarkers for disease monitoring and response are ongoing. Funding: ADC Therapeutics SA; writing: CiTRUS Health Group. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cell Mass and Stiffness As Integrative Biomarkers of Cell State in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) manifests extensive clinical phenotypic heterogeneity, ranging from indolent to aggressive, rapidly progressive disease. Molecular, genomic, transcriptomic, and immunophenotypic variation, as well as the presence of multiple subclones at diagnosis reflect the clinical diversity seen among MCL patients and make it challenging to define therapies that optimally align with individual patient risk. Biophysical phenotypes of cells, such as mass, density and stiffness, are valuable markers of changes in their intrinsic properties and closely associated with cellular states and functions, which have proven useful in elucidating fundamental processes in cell growth and development. We integrate single-cell biophysical features, which provide more granularity than canonical immunophenotypes, to further refine the biological characterization of MCL with the goal of defining rapid ex vivo biomarkers of clinical response or resistance. Methods: We utilize a device called suspended microchannel resonator (SMR) to perform biophysical measurements (mass, stiffness and density) of individual cells. We characterized the biophysical properties of MCL cells from representative sites, including peripheral blood, bone marrow and lymph nodes, of human clinical specimens and, additionally, spleen from six patient-derived xenografts (PDX) models of MCL. Furthermore, to identify genes that covary with lymphoma cell biophysical heterogeneity, we linked the biophysical measurements of individual MCL cells from the matched spleen, peripheral blood, bone marrow, and liver tissues of three carefully curated PDX models of MCL to downstream single-cell RNA-sequencing (SMR-scRNA-Seq). Results: We observed inter- and intra-tumor heterogeneity in mass and stiffness within each lymphoma specimen. Interestingly, these biophysical features reflect underlying histopathologic phenotypes, with higher mass and higher stiffness measured in blastoid and pleomorphic diseases as opposed to the smaller classical variant. By coupling biophysical measurement with transcriptomic profiling at single-cell resolution, we found that mass is strongly associated with the expression of genes annotated for cell division, cell cycle, and B-cell activation such as CCNB1, CDK1, CENPF, BLK, CD79A. Similarly, cell stiffness correlated with B-cell activation signatures and B-cell receptor signaling pathway activity, especially in the highly proliferative models. Functional genomics assays are ongoing to validate the contribution of these target pathways to MCL biophysical changes. To further define how B-cell activation impacts cellular biophysical properties, we profiled the biophysical properties of normal mature B-cell developmental stages from naïve B-cells to plasma cells. We found that cells at each differentiation stage possess a unique combination of mass, stiffness, and density. Highlighting the dynamic nature of these properties, B-cells undergo significant biophysical change after activation, with buoyant mass quadrupling upon activation and then rapidly decreasing by half. Preliminary data further reveal that mature B-cells lose approximately 25% of their stiffness after activation before reverting to their original stiffness after immunoglobulin class switching and even increasing in stiffness at the plasma cell stage. Most MCL cells are conventionally thought to derive from naïve pre-germinal center B cells, but our analysis revealed biophysical profiles overlapping strongly with more mature B cell states, suggesting that lymphomagenesis may entail reprogramming the machinery regulating cell mass and stiffness. We are now generating biophysical, transcriptomic, and genomic measurements within primary specimens from patients with MCL undergoing combined therapy targeting BTK, BCL2, and CD20 to assess their potential for inclusion within integrated predictive biomarkers. Conclusions: We established the biophysical landscape of MCL and non-malignant B-lymphoid cells to sharpen biological characterization and resolve tumor heterogeneity. We did so using a unique single-cell platform integrating transcriptomic and biophysical features as rapid, quantitative, and label-free measurements of live-cell state and phenotypic composition in pursuit of functionally integrated predictive biomarkers.

Usefulness of circulating EPAC1 as biomarkers of therapeutic response to GLP-1 receptor agonists.

The response to Glucagon-like peptide-1 receptor agonists (GLP-1RAs) is highly varia-ble among patients. Thus, the identification of predictive biomarkers of therapeutic response to GLP-1 RA could help us to optimize the use of this class of drugs. GLP-1RAs increase exchange proteins directly activated by cAMP (EPAC). The aim of the present study was to assess whether the increase of EPAC1 after GLP-1RAs treatment could be a biomarker of clinical response. After showing that GLP-1 (10ng/mL) significantly increased the expression of EPAC1 in human endo-thelial vascular cells (HUVEC), a pilot clinical study was planned. For this purpose 49 patients with type 2 diabetes who started treatment with liraglutide were included. EPAC1 concentration was determined by ELISA before and at one month of liraglutide treatment. We found that serum concentration of EPAC1 increased significantly after treatment with liraglutide. Only in those patients in whom EPAC1 increased (64%), a significant decrease in HbA1c, LDL-C, body mass index (BMI), and waist circumference was shown. This pilot study suggests that the increase of circulating EPAC1 after GLP-1RAs treatment could be a useful biomarker to predict clinical GLP1-RAs response.

Novel pathophysiological insights in autoimmune myasthenia gravis.

This review summarizes recent insights into the immunopathogenesis of autoimmune myasthenia gravis (MG). Mechanistic understanding is presented according to MG disease subtypes and by leveraging the knowledge gained through the use of immunomodulating biological therapeutics. The past two years of research on MG have led to a more accurate definition of the mechanisms through which muscle-specific tyrosine kinase (MuSK) autoantibodies induce pathology. Novel insights have also emerged from the collection of stronger evidence on the pathogenic capacity of low-density lipoprotein receptor-related protein 4 autoantibodies. Clinical observations have revealed a new MG phenotype triggered by cancer immunotherapy, but the underlying immunobiology remains undetermined. From a therapeutic perspective, MG patients can now benefit from a wider spectrum of treatment options. Such therapies have uncovered profound differences in clinical responses between and within the acetylcholine receptor and MuSK MG subtypes. Diverse mechanisms of immunopathology between the two subtypes, as well as qualitative nuances in the autoantibody repertoire of each patient, likely underpin the variability in therapeutic outcomes. Although predictive biomarkers of clinical response are lacking, these observations have ignited the development of assays that might assist clinicians in the choice of specific therapeutic strategies. Recent advances in the understanding of autoantibody functionalities are bringing neuroimmunologists closer to a more detailed appreciation of the mechanisms that govern MG pathology. Future investigations on the immunological heterogeneity among MG patients will be key to developing effective, individually tailored therapies.

Circulating miR-1246 and miR-485-3p as Promising Biomarkers of Clinical Response and Outcome in Melanoma Patients Treated with Targeted Therapy.

Simple SummarySeveral therapeutic options exist for patients with advanced BRAF-mutant melanoma. Biomarkers able to identify patients with refractory disease or with poor progression-free survival (PFS) and overall survival (OS) expectancy with respect to specific treatments might allow a more personalized therapeutic approach. Here, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan–Meier and univariate and multivariate Cox regression analyses were performed on miR-1246 and miR-485-3p baseline levels to assess their ability to discriminate between responding and non-responding patients and to determine their prognostic value for PFS and OS. Globally, our results suggest that circulating miR-1246 and miR-485-3p could be valuable biomarkers for identifying patients most likely to be resistant to targeted therapy or with a poor expectancy of PFS and OS. Prospective studies in a larger cohort of patients are warranted.Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan–Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.

Blood Immune Cell Biomarkers in Lung Cancer Patients Undergoing Treatment with a Combination of Chemotherapy and Immune Checkpoint Blockade.

Simple SummaryTumor cells can evade destruction via immune cells by expressing coinhibitory membrane molecules, which can suppress tumor-specific T cells. Immune checkpoint inhibitor therapies act by blocking these inhibitory pathways via monoclonal antibodies. Although this type of immunotherapy has shown promising results for advanced cancers of different entities during recent years, an important challenge is to identify the baseline characteristics of patients who will mostly benefit from such treatment. Blood biomarkers have limitations to reflect the tumor microenvironment but are easier to handle than markers in tumor lesions. The aim of our study was to identify blood cell parameters correlating with patients’ survival in 90 patients with non-small cell lung cancer undergoing immune checkpoint inhibitor therapy combined with chemotherapy. We found that patients with a neutrophil-to-lymphocyte ratio ≥6.1, a percentage of HLA-DRlow monocytes ≥22%, a frequency of slan+ non-classical monocytes <0.25%, and/or of dendritic cells ≤0.14% of leukocytes had a poorer prognosis. Long-term survivors were patients without any of the risk factors investigated. Our results implicate that blood neutrophil counts, special types of monocytes, and the number of blood dendritic cells might be useful predictive biomarkers for cancer patients’ survival.Although immune checkpoint inhibitor (ICI) therapies have improved the treatment of patients with advanced non-small cell lung cancer (NSCLC), several patients do not achieve durable clinical responses. Biomarkers for the prediction of therapy responses are urgently needed. To identify blood cell parameters correlating with patients’ survival, immune cells from 90 patients with NSCLC undergoing a combination of ICI and chemotherapy were prospectively monitored. At the time point of the first and third antibody administration, complete leukocyte blood count, the percentage of HLA-DRlow monocytes, the percentage of 6-Sulfo LacNAc (slan)+CD16+ non-classical monocytes, and the number of circulating dendritic cell (DC) subtypes, as well as T-, B-, and NK cells were determined by multi-color flow cytometry in peripheral blood. The prognostic value of the immune cell parameters investigated was evaluated by patients’ survival analysis, with progression-free survival (PFS) as the main criterion. A total of 67 patients (74.4%) showed a partial remission or a stable disease, and 35% of patients even survived 12 months and longer. Patients with a neutrophil-to-lymphocyte ratio (NLR) ≥6.1, a frequency of HLA-DRlow monocytes ≥22%, of slan+ non-classical monocytes <0.25% of leukocytes, and/or a sum of myeloid DC (MDC) and plasmacytoid DC (PDC) ≤0.14% of leukocytes had a poorer prognosis. The hazard ratio for PFS was 2.097 (1.208–3.640) for the NLR, 1.964 (1.046–3.688) for HLA-DRlow monocytes, 3.202 (1.712–5.99) for slan+ non-classical monocytes, and 2.596 (1.478–4.56) for the MDC/PDC sum. Patients without any of the four risk factors showed the best PFS. Furthermore, low NK cell counts correlated with shorter PFS (cutoff 200 cells/µL). Female patients had lower baseline NK cell counts and a shorter PFS. Our study confirms the usefulness of blood immune cells as biomarkers for clinical response and survival in NSCLC patients undergoing a combined ICI/chemotherapy.

Epidemiological and Clinical Factors Affecting the Response to Etanercept Among Patients with Rheumatoid Arthritis

Background: This study aimed to inspect the association between the response to etanercept among patients with rheumatoid arthritis and several epidemiological and clinical variables and medication adherence, as measured by medication possession ratio (MPR).&#x0D; Methods: A cohort study that enrolls 120 active rheumatoid arthritis patients. The baseline values of disease activity score for 28 joints (DAS28) score,erythrocyte sedimmintation rate (ESR), WBC, tender joints count (TJC), swelling joints counts (SJC), and medication adherence, as measured by medication possession ratio (MPR), were identified. All patients received etanercept treatment for three months, and then the clinical response to etanercept was assessed after the end of the three months duration. Factors affecting clinical response were evaluated by univariate and multivariate logistic regression analysis. The predictive performance of a single independent predictor was then assessed using a receiver operating characteristic (ROC) curve.&#x0D; Results: The results of the univariate logistic regression model showed that the smoking, disease duration, baseline DAS28, and MPR could predict the patients' proclivity for being non-responder. The multivariate logistic regression model showed that only baseline DAS28 (P&lt; 0.0001, OR=32.239, 95%CI: 4.941–210.338) and MPR (P=0.002, OR=0.00063, 95%CI: 0.00001–0.032) were independent predictive factors for the tendency of patients to be non-responder. ROC curve analysis disclosed that baseline ESR and DAS28 have a good area under the curve (AUC) with the optimal cut-off for the baseline ESR threshold was 52 mm/hr., whereas the baseline DAS28 threshold was 5.79.&#x0D; Conclusion: Current smoking is the main epidemiological factor that can predict the tendency for being non-responder. The potential of baseline ESR and DAS28 values as biomarkers for clinical response to etanercept in RA patients was identified by Receiver operating characteristic(ROC) analysis.

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity.

Abstract CT541A: Oncolytic viral reshaping of the tumor microenvironment to promote CAR T cell therapy for glioblastoma

Abstract The combination of chimeric antigen receptor (CAR) T cell therapy, which delivers large numbers of tumor reactive T cells, and oncolytic viral therapy, causing activation of host immune responses, is an attractive approach for improving outcomes for patients with glioblastoma (GBM). Here we present data from two independent phase I clinical trials evaluating IL13Rα2-targeted CAR therapy (NCT02208362) and C134 oncolytic viral (OV) therapy (NCT03657576) for the treatment of recurrent GBM (rGBM), along with preclinical studies supporting the utility of combining these two therapies. For NCT02208362, locoregional delivery of IL13Rα2-targeted CAR T cells were evaluated in heavily pretreated patients with rGBM. Interrogating biomarkers of clinical response revealed that levels of intratumoral T cells prior to treatment were positively associated with overall survival; furthermore, two patients who achieved a complete response had the highest levels of intratumoral CD3+ T cells pre-therapy. These findings suggest that therapeutic strategies which increase endogenous immune infiltrates could augment CAR T cell mediated responses. For NCT03657576, intratumoral delivery of C134, a herpes simplex virus (HSV-1) that has been genetically engineered to safely replicate and kill glioma tumor cells, is also being evaluated for treatment of rGBM. We report findings from a patient treated intratumorally with 1 × 106 pfu of C134. At 6-7 weeks post treatment this patient had MRI changes that suggested possible recurrence or pseudoprogression, and therefore underwent resection with biopsy assessment. Evaluation of virus-treated areas showed increased immune infiltrates as compared to untreated tumor sites, suggesting that C134 activated host immune responses. These clinical findings provide the rationale for evaluating a combination therapy of C134 OV and IL13Rα2-CAR T cells to potentially reshape the tumor microenvironment (TME) and enhance CAR therapy. In orthotopic GBM models in nude mice, we show that co-treatment with the two agents gave no adverse reaction, and more notably pre-treatment with C134 re-shaped the TME by increasing immune cell infiltrates and enhanced the efficacy of sub-therapeutic doses of CAR T cell therapy delivered either intraventricularly or intratumorally. Ongoing preclinical studies aim to provide detailed phenotypic analysis, as well as a mechanistic understanding of this combination approach to support the potential benefit of a soon to be opened combination trial evaluating C134 and IL13Rα2-CAR T cells. In this clinical trial in patients with IL13Rα2+ rGBM and anaplastic astrocytoma, increasing doses of intratumorally administered C134 will be followed by dual intracranial intratumoral and intraventricular administration of IL13Rα2-targeted CAR T cell therapy. Citation Format: Christine E. Brown, Agata Xella, Jonathan C. Hibbard, Vanessa Salvary, Brenda Aguilar, Jamie Wagner, Bruce Dezube, Knut Niss, Lynn Bayless, James Edinger, Jianmei Leavenworth, Stephen J. Forman, Behnam Badie, James M. Markert, Kevin A. Cassady. Oncolytic viral reshaping of the tumor microenvironment to promote CAR T cell therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT541A.

Abstract LB107: Novel transcriptional signatures associated with antitumor activity in vidutolimod (vidu)-treated patients (pts) with anti-PD-1-refractory melanoma and non-small cell lung cancer (NSCLC)

Abstract Background: Vidu is a first-in-class CpG-A TLR9 agonist in a virus-like particle that activates plasmacytoid dendritic cells (pDC), thus bridging innate and adaptive immunity. Intratumoral (IT) vidu alone or in combination with intravenous (IV) anti-PD-(L)1 has shown evidence of antitumor activity in pts with anti-PD-(L)1-refractory melanoma or NSCLC. As biomarkers associated with anti-PD-(L)1 response were not predictive of vidu activity, we sought to identify novel transcriptional signatures. Methods: RNA-Seq was performed on baseline biopsies from pts with anti-PD-(L)1-refractory melanoma (NCT02680184; RECIST v1.1 responders [R; n=20]; nonresponders [NR; n=78; 30 pts with stable disease + 48 pts with progressive disease [PD]]) or NSCLC (NCT03438318; N=11) treated with vidu ± anti-PD-(L)1. IFNg18 signature was used to characterize melanomas as non-T cell-inflamed (non-Tinfl), T cell-inflamed (Tinfl), or intermediate. Gene Set Enrichment Analysis (GSEA) using MSigDB and other signatures (&amp;gt;19,000) was performed on non-Tinfl melanoma in R vs NR (false discovery rate [FDR] &amp;lt;0.25). Deconvolution of immune cells was performed using TIMER2.0. Prediction models were generated using QLattice. Signatures were characterized using both publicly available bulk and single-cell (sc) RNA-Seq datasets of pDC subsets or PD-1/CTLA-4 blockade response datasets. Results: GSEA on non-Tinfl melanoma biopsies revealed 2 gene signatures (COPII vesicle and Golgi targeting) most strongly associated with R (FDR &amp;lt;0.16). Leading edge analysis of these signatures identified 35 common core genes (CC) that strongly differentiated RECIST 1.1 R vs PD. As independent validation, CC enrichment was also significantly associated with R in intermediate melanoma (p=0.009) and with tumor shrinkage in NSCLC (p=0.027). CC was significantly associated with R to vidu single-agent or combination treatment, but not with clinical baseline prognostic factors or IFNg18. In Tinfl melanoma, CC was not associated with R, but myeloid signatures were significantly associated with NR. A model based on CC and transcription factor ELF2 predicted R in melanoma (AUC 0.93 [95% CI 0.82-1.00]). In public datasets, CC was not associated with R to PD-1/CTLA-4 blockade, but CC was highly expressed in the type I interferon-secreting subset of pDCs and some myeloid cells. CC expression was prevalent in most tumor types and was independent of IFNg18 in TCGA. Conclusion: In pts with anti-PD-1-refractory melanoma or NSCLC, transcriptional signatures of COPII vesicle and Golgi targeting (functionally related to TLR9 activation) were associated with antitumor activity of IT vidu ± IV anti-PD-(L)1. Ongoing bulk and sc RNA-Seq analyses may clarify the underlying biology and the signature’s potential role as a predictive biomarker for clinical response to vidu. Citation Format: Hong Liu, Luping Zhao, Ping Zheng, Riyue Bao, Jason J. Luke, Marcelo V. Negrao, Shakoora A. Sabree, George J. Weiner, Sujatha Kumar, Dmitri Bobilev, James E. Wooldridge, Arthur M. Krieg. Novel transcriptional signatures associated with antitumor activity in vidutolimod (vidu)-treated patients (pts) with anti-PD-1-refractory melanoma and non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB107.