Immunophenotypic Analysis Research Articles

Effect of propofol and isoflurane on NK Cells: A pilot study on perioperative breast cancer patients from Eastern India

Introduction: In onco-anesthesiology, the selection between intravenous and volatile anesthetics for maintaining NK cell response during cancer surgery has been an ambiguous area. In the absence of any such comparative Indian report, this pilot study aimed to investigate the effect of intravenous anesthetic, Propofol, and volatile anesthetic, Isoflurane on the NK cell-mediated immune-inflammatory response of perioperative breast cancer patients. Materials and Methods: Treatment naive breast cancer patients (N, 50) selected for surgery were randomly subjected to either Isoflurane (N, 25) or Propofol (N, 25) anesthetic group. The peripheral blood samples were collected 1 day before surgery (Pre), 1 h after incision (Intra) and 48 h after surgery (Post). The blood samples were subjected to immunophenotyping, activation, degranulation, and cytokine analysis. Results: The frequency of NK cell populations did not differ between the Isoflurane and Propofol groups irrespective of cancer stage II/III. NK cell degranulation marker CD107a and activation marker CD335 expression were significantly reduced in the postoperative period compared to the preoperative group in the Isoflurane group but Propofol did not inflict such alterations. Both Isoflurane/Propofol elicited similar trends in the cytokine profiles of interleukin (IL)-6, IL-15, interferon (IFN)-g and transforming growth factor (TGF)-β levels. Isoflurane significantly increased IL-8 during the intraoperative period and matrix metalloproteinase (MMP)-9 during the postoperative period whereas no such changes were observed with Propofol. Conclusion: This pilot study indicated that Propofol partially proved better over Isoflurane in maintaining the activation (CD335) and degranulation of NK cells (CD107a) and alleviating inflammation (IL-8 and MMP-9) during the perioperative period in the breast cancer patients.

Differentiation of neural stem cells from human olfactory mucosa into dopaminergic neuron-like cells.

The aim of this study was to develop an alternative treatment method for neurodegenerative diseases with dopaminergic neuron loss such as Parkinson's disease by differentiating cells obtained from human olfactory mucosa-derived neural stem cells (hOM-NSCs) with neurotrophic agents in vitro. hOM-NSCs were isolated and subjected to immunophenotypic and MTT analyses. These hOM-NSCs were then cultured in a 3D environment to form neurospheres. The neurospheres were subjected to immunophenotypic analysis and neuronal differentiation assays. Furthermore, hOM-NSCs were differentiated into dopaminergic neuron-like cells in vitro. After differentiation, the dopaminergic neuron-like cells were subjected to immunophenotypic (TH, MAP2) and genotypic (DAT, PITX3, NURR1, TH) characterization. Flow cytometric analysis showed that NSCs were positive for cell surface markers (CD56, CD133). Immunofluorescence analysis showed that NSCs were positive for markers with neuronal and glial cell characteristics (SOX2, NESTIN, TUBB3, GFAP and NG2). Immunofluorescence analysis after differentiation of hOM-NSCs into dopaminergic neuron-like cells in vitro showed that they were positive for a protein specific for dopaminergic neurons (TH). qRT-PCR analysis showed that the expression of dopaminergic neuron-specific genes (DAT, TH, PITX3, NURR1) was significantly increased. It was concluded that hOM-NSCs may be a source of neural stem cells that can be used for cell replacement therapies in neurodegenerative diseases such as Parkinson's disease, are resistant to cell culture, can differentiate into neuronal and glial lineage, are easy to obtain and are cost effective.

Artificial intelligence and classification of mature lymphoid neoplasms.

Hematologists, geneticists, and clinicians came to a multidisciplinary agreement on the classification of lymphoid neoplasms that combines clinical features, histological characteristics, immunophenotype, and molecular pathology analyses. The current classification includes the World Health Organization (WHO) Classification of tumours of haematopoietic and lymphoid tissues revised 4th edition, the International Consensus Classification (ICC) of mature lymphoid neoplasms (report from the Clinical Advisory Committee 2022), and the 5th edition of the proposed WHO Classification of haematolymphoid tumours (lymphoid neoplasms, WHO-HAEM5). This article revises the recent advances in the classification of mature lymphoid neoplasms. Artificial intelligence (AI) has advanced rapidly recently, and its role in medicine is becoming more important as AI integrates computer science and datasets to make predictions or classifications based on complex input data. Summarizing previous research, it is described how several machine learning and neural networks can predict the prognosis of the patients, and classified mature B-cell neoplasms. In addition, new analysis predicted lymphoma subtypes using cell-of-origin markers that hematopathologists use in the clinical routine, including CD3, CD5, CD19, CD79A, MS4A1 (CD20), MME (CD10), BCL6, IRF4 (MUM-1), BCL2, SOX11, MNDA, and FCRL4 (IRTA1). In conclusion, although most categories are similar in both classifications, there are also conceptual differences and differences in the diagnostic criteria for some diseases. It is expected that AI will be incorporated into the lymphoma classification as another bioinformatics tool.

Sex-dependent responses to high concentration of binge ethanol in spleen of adolescent F344 rats.

We previously reported that binge ethanol induces atrophy of the spleen, a key immune organ, in adolescent male F344 rats. Because there are significant sex effects in immune function, we investigated whether binge ethanol exerts sex-dependent effects on the spleen, including producing splenic atrophy. We gave F344 rats ethanol (4.8 g/kg/day; 52% w/v; i.g.) on postnatal days [PND] 36 ~ 38 and sacrificed them on PND 39 for spleen collection. We performed immunophenotyping analysis of splenic cells and examined the expression of 158 genes related to alcohol metabolism, epigenetic modification, and immune regulation in the spleens of adolescent (PND 39) male and female rats. Following a 3-day ethanol exposure, a loss of body weight, and absolute and relative spleen weight, was seen only in male adolescent rats. Ethanol altered the relative proportions of lymphocyte subtypes in both sexes with different patterns. We also found that 3-day ethanol exposure induced sex-dependent gene expression changes in spleen. Among the 158 genes studied, the expression of only three genes was significantly increased in female rats. However, the expression of 30 genes was significantly increased/decreased in male rats. Female rats had greater expression of alcohol metabolizing enzyme genes in the spleen under physiological conditions and when stimulated by binge ethanol. The genes are involved in epigenetic modification were differentially expressed in a sex-dependent manner. We found that male adolescent rats were more sensitive to binge ethanol than female rats. Differential expression of the genes related to alcohol metabolism and epigenetic modification (of DNA methyltransferase and histone deacetylases) between the sexes could account for the observed sex-dependent responses to binge ethanol in adolescent rats.

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [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 440.

Abstract 2879: Spatiotemporal fractionation: An innovative concept of fractionated radiotherapy

Abstract Impressive outcomes in therapeutic radiation oncology have been achieved in the past decade through the development of advanced computer technology and imaging methods, such as intensity modulated radiotherapy. Ideally, radiotherapy treatment delivers a high dose to the tumor while sparing the surrounding healthy tissues and organs at risk from radiation damage. Spatiotemporal Fractionated Radiotherapy has emerged as an innovative technique that delivers different dose distributions to different parts of the tumor in order to achieve high doses to the target while ensuring a low dose-bath in the surrounding normal tissue. Spatiotemporally fractionated regimens showed in silico up to 10 - 15% sparing of normal tissue from radiation damage, while ensuring isoeffective tumor control compared to conventionally fractionated regimens. However, prior to advancing spatiotemporal fractionation to the clinical level, it is important to probe the validity of the main assumption of spatiotemporal fractionation derived from the Biologically Effective Dose (BED) model: two different fractionation schemes that implement a high and a low dose will lead to equivalent clinical effects, regardless of dose-treatment sequence. A tumor growth delay study was performed in a subcutaneous immunocompetent murine tumor model (MC38-tumor cell derived) receiving two different fractionation regimens. Particularly, tumors belonging to the two treatment groups received the same dose combination - a high (12 Gy) and a low (6 Gy) dose fraction separated by 72 hours - with the doses delivered in opposite order in the two treatment groups. The same experimental outline was performed in a subcutaneous immunodeficient murine tumor model derived from the same tumor cells. Tumors were also characterized by immunophenotyping analysis seven days after first irradiation. Even though the two regimens deliver the same physical dose to the tumor - and would be considered equivalent according to the BED model - different tumor growth delays were observed in a time window of 30 days after first irradiation depending on the treatment group. Such differences were observed in the immunocompetent model but not in the immunodeficient model. The immunophenotyping results showed immunostimulatory vs immunosuppressive phenotypes of the tumor microenvironment depending on the order of the treatment doses. An additional tumor growth delay study including immune checkpoint inhibitors confirmed the fundamental role of the immune system with the differential efficacy outcomes of the two regimens. Overall, these intriguing results suggest that a differential radiotherapy-induced immune response to different doses of ionizing radiation plays an important role in the treatment outcome and asks for re-evaluation of the BED model taking scheduling-dependent parameters into consideration. Citation Format: Irene Vetrugno, Irma Telarovic, Nathan Torelli, Jan Unkelbach, Martin Pruschy. Spatiotemporal fractionation: An innovative concept of fractionated 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 2879.

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [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 534.

Abstract 4907: RanBALL: Identifying B-cell acute lymphoblastic leukemia subtypes based on an ensemble random projection model

Abstract As the most common pediatric malignancy, B-cell acute lymphoblastic leukemia (B-ALL) has multiple distinct subtypes characterized by recurrent and sporadic somatic and germline genetic alterations like chromosomal alteration, transcription factor rearrangement or kinase inhibition. The treatment of B-ALL patients is personalized based on specific subtypes, as the treatment responses for different B-ALL subtypes may vary considerably. Identification of B-ALL subtypes can facilitate risk stratification and enable tailored therapeutic approaches. Existing methods for B-ALL subtyping primarily depend on immunophenotypic, cytogenetic and genomic analyses, which would be costly, complicated, and laborious in clinical practice applications. To overcome these challenges, we present RanBALL (an Ensemble Random Projection-Based Model for Identifying B-Cell Acute Lymphoblastic Leukemia Subtypes), an accurate and cost-effective model for B-ALL subtype identification based on transcriptomic profiling only. RanBALL leverages random projection (RP) to construct an ensemble of dimension-reduced multi-class classifiers for B-ALL subtyping. Specifically, the transcriptomic profiling features were projected onto low-dimensional spaces by random projection matrices whose elements conform to a distribution characterized by zero mean and unit variance. To ensure reliable and robust performance, we selected 20 subspace dimensions ranging from 600 to 2500, with intervals of 100. The transformed low dimensional data matrix was used for training an ensemble of multi-class support vector machine (SVM) classifiers, each corresponding to one of the RP matrices of various dimensions. The predicted probabilistic scores of each B-ALL subtype were integrated for determining the final decision. Results based on 10 times 10-fold cross validation tests for >1700 B-ALL patients demonstrated that the proposed model achieved an accuracy of 93.7%, indicating promising prediction capabilities of RanBALL for B-ALL subtyping. Furthermore, the 30% held-out tests suggested that the model was robust and consistent to maintain high confidence levels for accurate predictions. The high accuracies of RanBALL suggested that our model could effectively capture underlying patterns of transcriptomic profiling for accurate B-ALL subtype identification. To extend the impact of RanBALL, we have established a free and publicly available python package for RanBALL available at https://github.com/wan-mlab/RanBALL. We believe RanBALL will facilitate the discovery of B-ALL subtype-specific marker genes and therapeutic targets, and eventually have consequential positive impacts on downstream risk stratification and tailored treatment design. Citation Format: Lusheng Li, Hanyu Xiao, Joseph D. Khoury, Jieqiong Wang, Shibiao Wan. RanBALL: Identifying B-cell acute lymphoblastic leukemia subtypes based on an ensemble random projection model [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 4907.

Abstract 3648: The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL

Abstract Point process analyses are widely used in ecology and geography to understand the impact and relevance of spatial distribution of points in a complex system. To date, only few studies have explored point process analyses in the context of tumor heterogeneity. Malignancies often show variable patterns of cellular distribution, and the relationship of these topographic variables with underlying biological processes and clinical outcomes is not well understood. Here, we performed spatial point process analyses in diffuse large B-cell lymphoma (DLBCL), leveraging advances in multiplexed immunohistochemistry and cellular phenotyping, with downstream multi-omic and clinicopathological analyses. We have demonstrated that survival in DLBCL is strongly associated with the fraction of malignant cells showing co-expression of the oncogenes MYC and BCL2 in the absence of BCL6 (M+2+6-). These cells display non-random spatial organization within tumor infiltrates, deviating from a poisson distribution. We therefore modeled the spatial organization of M+2+6- cells within DLBCL, using x-y coordinate information from multiplexed fluorescent immunohistochemistry (mfIHC) in four independent cohorts (N=449 patients). We derived spatial point patterns, upon which Geyers point process models were applied. We observed that patients could be divided into two groups based on these: one group showed “clustered” spatial organization, while the other displayed a “dispersed” M+2+6- cell distribution. Cases with predominantly “dispersed” M+2+6- cells showed shorter overall survival in all analyzed cohorts (P < 0.05 in 4/4 cohorts). We performed multi-omic analyses to identify potential biological explanations for this association between topography and clinical outcome. Using digital spatial profiling of the transcriptome from CD20, CD3 and CD68 compartments in 47 DLBCL samples, we observed that lymphomas with an M+2+6- ‘dispersed’ phenotype had an immunologically cold microenvironment enriched in Tregs and exhausted CD4+ and CD8+ T cells. Through an integration with single-cell transcriptomic analyses (N=22 samples) we observed that malignant B-cells from cases with M+2+6- ‘dispersed’ phenotype expressed genes implicated in cell migration and adhesion, but also in potentially targetable immune checkpoints such as LAG3. In summary, by integrating multiplexed immunophenotyping and point process analysis with molecular profiling in the setting of DLBCL, we show that a dispersed pattern of distribution of the M+2+6- subclonal fraction uniquely confers poor prognosis, and associates with a potentially reversible immune cold microenvironment composition. This represents the first demonstration that the spatial distribution of malignant cell subpopulations in lymphoma can embody biological and clinical significance. Citation Format: Shruti Shridar, Michal Marek Hoppe, Patrick Jaynes, Gayatri Kumar, Siddham Jasoria, Ziwei Meng, Vaibhav Rajan, Kasthuri Kannan, Claudio Tripodo, Anand Devaprasath Jeyasekharan. The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL [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 3648.

Abstract 3863: Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies

Abstract Flow cytometry assays using fresh whole blood which are interrogating either an extremely rare cell subset or a depleting immunotherapy, may require samples to be processed from global clinical sites over a wide geographical area, which can pose a major challenge for clinical operations. Precision for Medicine (PfM) has addressed these operational challenges using commercial stabilizers which are added to the blood allowing samples to be frozen at -80°C and subsequently batch processed for sample testing. We have performed a fit for purpose assay qualification of a multiparameter flow cytometry panel allowing the immunophenotypic analysis of monocyte, T cell, B cell and NK immune populations using Cytodelics™ fixed/stabilized healthy donor whole blood. Additionally, the assay was verified in whole blood samples from multiple solid tumor disease states including melanoma, colorectal cancer, head & neck, non-small cell lung cancer to demonstrate clinical application. In these oncology samples, we were able to assess both immune cell frequencies and their activation status in both fresh whole blood as well as fixed whole blood over a short-term sample stability study, where the samples had been stored at -80°C for several months. This data provides a representative example of the use of commercial stabilizing fixatives for clinical studies to extend the stability window of whole blood for subsequent assessment by flow cytometry. Fixed whole blood can be stored at -80°C, shipped on dry ice allowing multiple timepoints in a cohort to be assessed together, thus minimizing assay variability as compared to fresh whole blood analysis, and mitigating the impact of shipping delays and temperature excursions on the samples. With sufficient time for assay development there are several commercial stabilizers that can be tested to determine if the markers in the flow assay are compatible with these reagents, providing a valuable option to assess the impact of immunotherapies on the immune cells of interest and reducing operational challenges in global studies. Citation Format: Jackie Benko, Samantha Splitt, Angelina Bisconte, Laura Lozza, Rachel Owen, Deborah Phippard. Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies [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 3863.

Abstract 581: Identification of potential biomarkers of response to RASMULTI(ON) plus anti-PD-1 combination in preclinical PDAC models

Abstract Mutant KRAS is present in more than 80% pancreatic cancer, promotes cancer cell survival, and protects cancer cells from immune recognition and attack. We have shown that inhibitors of the active state of RAS (RAS(ON)) promote cancer neoantigen recognition and synergize with immunotherapy in preclinical immunogenic models. 1 We evaluated the impact of RMC-6236, an investigational RASMULTI(ON) inhibitor on anti-tumor activity and tumor microenvironment (TME) remodeling in two congenic KRASG12D/+ GEMM-derived PDAC models resistant to anti-PD-1 therapy - KPCY clone 2 (6694c2) and clone 3 (2838c3), developed by Ben Stanger at UPenn. 2 Daily oral administration of RMC-6236 drove tumor regressions in KPCY clone 3, leading to 10% durable complete responses (CRs). In combination with anti-PD-1, 40% of durable CRs were achieved. However, RMC-6236 only drove tumor growth inhibition in KPCY clone 2, and no combination benefit with anti-PD-1. We conducted preclinical biomarker analyses in the TME and periphery to explore differences in response to RMC-6236 alone and in combination with anti-PD-1 in these models. Assessment of baseline predictive biomarkers in tumors elucidated that the KPCY clone 3 cells developed tumors with lower suppressive myeloid cells (gMDSCs) and higher CD8+ T cell infiltration compared to those derived from KPCY clone 2 cells. IHC analyses showed that T cells in clone 3 were in the tumor core, while CD8+ T cells in clone 2 were at the tumor border (the latter frequently observed in PDAC patients). RMC-6236 induced transformation of the TME in favor of anti-tumor immunity in only the KPCY clone 3 - with decreased immunosuppressive gMDSCs, and increased T cells. Limited modulation of the TME was observed in the KPCY clone 2 cells-derived tumors, and gMDSC levels significantly higher than those in baseline tumors of KPCY clone 3. Peripheral immunophenotyping analyses revealed that baseline peripheral suppressive myeloid cells (mMDSCs) were significantly higher in mice harboring tumors originated from KPCY clone 2 cells, suggestive of a more systemic immunosuppressive status. Studies are ongoing to assess additional peripheral immune signatures of preclinical activity of RMC-6236 in combination with anti-PD-1. Taken together, baseline tumor levels of suppressive myeloid cells (gMDSCs) were shown to be a negative predictive biomarker for treatment outcome to RMC-6236 +/- anti-PD-1 in KRAS mutant preclinical PDAC models. Peripheral mMDSCs were shown to be higher in the mice with tumors from the PDAC model not sensitive to the combination of RMC-6236 with anti-PD-1, suggesting that it could be predictive of poor outcome for this combination. These studies in immune-competent models of RAS mutant PDAC allow us to generate tumor and blood-based biomarkers that may predict outcomes to RAS(ON) inhibitor combinations in the clinic. 1 Menard et al. AACR 2023 2 Li et al. Immunity 2018 Citation Format: Lillian Seu, Marie Menard, Jasmine Lee, Christopher Chow, Cristina Blaj, Mariela Moreno Ayala, Nataliya Shifrin, Avery Salmon, Swetha Ganesh, Philip Wig, Alice Kumamoto, Paola Soto-Perez, Tippy Taavili, Vivian Morton, James W. Evans, Jacqueline A. Smith, Elsa Quintana. Identification of potential biomarkers of response to RASMULTI(ON) plus anti-PD-1 combination in preclinical PDAC models [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 581.

Abstract 35: In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy

Abstract Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in immunotherapy, offering promising outcomes in the management of hematological malignancies. Current CAR-T cell in vitro manufacturing routes involve T cell isolation, purification, activation, gene transduction and cell expansion. The process duration and the costs present significant barriers. Therefore, there is a need for innovative strategies to address these concerns. Herein, we developed lipid nanoparticles (LNPs) to deliver CAR-mRNA efficiently and safely to T cells in vivo, thereby enabling in situ CAR-T cell engineering. By optimizing their composition and introducing anti-CD5 as the targeting ligand, we were able to enhance mRNA delivery and T cell transfection efficiency while maintaining T cell function. Furthermore, to augment T cell responsiveness to exogenous genetic elements, we investigated the pretreatment of T cells with interleukin (IL-7), thereby modulating T cell homeostasis by enhancing survival and proliferation, and rendering the cells more receptive to exogenous genetic modifications. We tracked firefly luciferase (FLuc) in naïve Balb/c mice using in vivo and ex vivo bioluminescence imaging after intravenous injection of anti-CD5 FLuc-mRNA LNPs. We observed positive mRNA expression in lymphoid organs, particularly in the spleen. Additionally, we harvested the lymphoid organs, and performed immunophenotyping analysis using flow cytometry after the administration of anti-CD5 LNPs encapsulating mRNA encoded CD19-CAR with a flag tag. We showed enhanced LNP binding in 65.2% of T cells and CAR expression in 11.64% of T cells as compared with non-treated controls, anti-CD5 FLuc-mRNA LNPs, or IgG CAR-mRNA LNPs. In a CD19+ A20 tumor-bearing mouse model, treatment with CAR-LNPs resulted in a significant increase in chemoattractants (CCL2, CCL4, CCL5, CCL7 and CXCL10) and pro-inflammatory cytokines (TNF-α, IFN-γ, IL6, IL8 and IL15) in blood, and infiltration of T cells and other immune cells in tumors, compared to the non-treated control group over 48 hours post treatment. In vivo engineering allows simultaneous CAR T cell engineering and delivery, reducing the time between manufacturing and treatment, minimizing contamination risks, eliminating complex ex vivo processes, and reducing the need for high-dose preconditioning chemotherapy. In conclusion, the adoption of in situ CAR-T cell production holds promise for improving this therapeutic approach. The authors acknowledge the funding support from NIHR01CA253316 and Leukemia and Lymphoma Society (LLS, Grant ID: 8022-20) Citation Format: Nisi Zhang, Elise Robinson, Angelie Rivera-Rodriguez, James Wang, Yutong Guo, Bo Wu, Spencer K. Tumbale, Marina Raie, Mohammed Inayathullah, Debra K. Czerwinski, Ronald Levy, Katherine W. Ferrara. In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy [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 35.

Characteristics of chronic lymphocytic leukemia in Japan: Comprehensive analysis of the CLLRSG-01 study.

Chronic lymphocytic leukemia (CLL) is rare in Japan. We conducted the nationwide, prospective observational study CLLRSG-01 to clarify the current state of CLL in Japan and to make accurate international comparisons by preparing naturally air-dried smears like those used in other countries. Of the 201 untreated patients enrolled and evaluated, 119 were diagnosed with CLL and 82 with non-CLL mature B-cell neoplasms, based on the WHO classification. Of the 119 CLL patients, 90 were classified as typical and 29 as atypical according to FAB classification morphology, with the proportion of atypical CLL consistent with reports from other countries. Immunophenotypic analyses by flow cytometry showed that 55% of Japanese CLL patients had a Matutes score of 4 or higher, which is lower than the rate of about 90% in Europeans. Mutated IGHV was identified in 80% of Japanese CLL patients, which is a higher rate than in Western patients. The most frequent IGHV gene was VH3-30 (15%), followed by VH3-23 (12%) and VH4-34 (10%). VH1-69, the most common gene in Western countries, was identified in only one patient. These results indicate that the pattern of immunophenotypes and IGHV gene usage in Japanese CLL patients differs from that in Western patients.

Immunophenotypic and Gene Expression Analyses of the Inflammatory Microenvironment in High-Grade Oral Epithelial Dysplasia and Oral Lichen Planus

BackgroundOral lichen planus (OLP) and oral epithelial dysplasia (OED) present diagnostic challenges due to clinical and histologic overlap. This study explores the immune microenvironment in OED, hypothesizing that immune signatures could aid in diagnostic differentiation and predict malignant transformation.MethodsTissue samples from OED and OLP cases were analyzed using immunofluorescence/immunohistochemistry (IF/IHC) for CD4, CD8, CD163/STAT1, and PD-1/PDL-1 expression. RNA-sequencing was performed on the samples, and data was subjected to CIBERSORTx analysis for immune cell composition. Gene Ontology analysis on the immune differentially expressed genes was also conducted.ResultsIn OED, CD8 + T-cells infiltrated dysplastic epithelium, correlating with dysplasia severity. CD4 + lymphocytes increased in the basal layer. STAT1/CD163 + macrophages correlated with CD4 + intraepithelial distribution. PD-1/PDL-1 expression varied. IF/IHC analysis revealed differential immune cell composition between OED and OLP. RNA-sequencing identified upregulated genes associated with cytotoxic response and immunosurveillance in OED. Downregulated genes were linked to signaling, immune cell recruitment, and tumor suppression.ConclusionsThe immune microenvironment distinguishes OED and OLP, suggesting diagnostic potential. Upregulated genes indicate cytotoxic immune response in OED. Downregulation of TRADD, CX3CL1, and ILI24 implies dysregulation in TNFR1 signaling, immune recruitment, and tumor suppression. This study contributes to the foundation for understanding immune interactions in OED and OLP, offering insights into future objective diagnostic avenues.

197MO Final outcome, immunophenotypic and biomarker analysis of intercalated avelumab plus platinum-based chemotherapy in patients with extensive-stage small cell lung cancer (PAVE): A pilot phase II study of the Hellenic co-operative oncology group

197MO Final outcome, immunophenotypic and biomarker analysis of intercalated avelumab plus platinum-based chemotherapy in patients with extensive-stage small cell lung cancer (PAVE): A pilot phase II study of the Hellenic co-operative oncology group

Pathological and imaging features, treatment, and prognosis of primary intraventricular lymphoma: A review of cases from a single center

PurposeThe purpose of this retrospective study was to analyze the imaging and pathological features, treatment, and prognosis of patients with primary intraventricular lymphomas (PIL) in order to enhance physicians' understanding of the diagnosis and treatment of PIL. MethodsA retrospective analysis was conducted on 13 cases of PIL that were hospitalized in our institution. Clinical and imaging data of the patients were collected and compared with the pathology data to summarize and analyze the qualitative diagnostic value of magnetic resonance (MR) features. ResultsAmong the enrolled patients, there were nine males and four females, with an average age of (56 ± 9.0) years. The major clinical features observed in PIL patients were headache and dizziness. All 13 patients underwent plain and contrast-enhanced MR scans, revealing multiple foci in 7 cases and single foci in 6 cases. The lesions were located in the lateral ventricle in 10 cases, the third ventricle in 4 cases, and the fourth ventricle in 4 cases. Plain MR scans demonstrated an isointense or slightly hypointense signal on T1-weighted imaging (T1WI) and an isointense or slightly hyperintense signal on T2-weighted imaging (T2WI). Contrast-enhanced scans showed uniform and consistent enhancement of the tumors. Surgical treatment was performed in all patients, and postoperative pathology confirmed the presence of diffuse large B-cell lymphoma. ConclusionsPIL exhibits specific imaging and pathological features, with diffuse large B-cell lymphoma being the main pathological type. Pathological examination and immunophenotype analysis serve as the gold standards for PIL diagnosis.

XIAP overexpressing inflammatory breast cancer patients have high infiltration of immunosuppressive subsets and increased TNFR1 signaling targetable with Birinapant

ObjectiveTo assess the expression pattern of X-linked inhibitor of apoptosis protein (XIAP), a cellular stress sensor, and delineate the associated changes in the tumor immune microenvironment (TiME) for prognostic value and new therapeutic targets in inflammatory breast cancer (IBC). MethodsImmunohistochemistry was conducted to assess the spatial localization of immune subsets, XIAP, and PDL1 expression in IBC and non-inflammatory breast cancer (nIBC) pretreatment tumors (n = 142). Validation and further exploration were performed by gene expression analysis of patient tumors along with signaling studies in a co-culture model. ResultsHigh XIAP in 37/81 IBC patients correlated significantly with high PD-L1, increased infiltration of FOXP3+ Tregs, CD163+ tumor-associated macrophages (TAMs), low CD8/CD163 ratio in both tumor stroma (TS) and invasive margins (IM), and higher CD8+ T cells and CD79α+ B cells in the IM. Gene set enrichment analysis identified cellular stress response- and inflammation-related genes along with tumor necrosis factor receptor 1 (TNFR1) expression in high-XIAP IBC tumors. Induction of TNFR1 and XIAP was observed when patient-derived SUM149 IBC cells were co-cultured with human macrophage-conditioned media simulating TAMs, further demonstrating that the TNF-α signaling pathway is a likely candidate governing TAM-induced XIAP overexpression in IBC cells. Finally, addition of Birinapant, a pan IAP antagonist, induced cell death in the pro-survival cytokine-enriched conditions. ConclusionUsing immunophenotyping and gene expression analysis in patient biospecimens along with in silico modeling and a preclinical model with a pan-IAP antagonist, this study revealed an interplay between increased TAMs, TNF-α signaling, and XIAP activation during (immune) stress in IBC. These data demonstrate the potential of IAP antagonists as immunomodulators for improving IBC therapeutic regimens.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Anoikis-related genes as potential prognostic biomarkers in gastric cancer: A multilevel integrative analysis and predictive therapeutic value.

Gastric cancer (GC) is a frequent malignancy of the gastrointestinal tract. Exploring the potential anoikis mechanisms and pathways might facilitate GC research. The authors aim to determine the significance of anoikis-related genes (ARGs) in GC prognosis and explore the regulatory mechanisms in epigenetics. After describing the genetic and transcriptional alterations of ARGs, we searched differentially expressed genes (DEGs) from the cancer genome atlasand gene expression omnibusdatabases to identify major cancer marker pathways. The non-negative matrix factorisation algorithm, Lasso, and Cox regression analysis were used to construct a risk model, and we validated and assessed the nomogram. Based on multiple levels and online platforms, this research evaluated the regulatory relationship of ARGs with GC. Overexpression of ARGs is associated with poor prognosis, which modulates immune signalling and promotes anti-anoikis. The consistency of the DEGs clustering with weighted gene co-expression network analysisresults and the nomogram containing 10 variable genes improved the clinical applicability of ARGs. In anti-anoikis mode, cytology, histology, and epigenetics could facilitate the analysis of immunophenotypes, tumour immune microenvironment (TIME), and treatment prognosis. A novel anoikis-related prognostic model for GC is constructed, and the significance of anoikis-related prognostic genes in the TIME and the metabolic pathways of tumours is initially explored.

Innate and adaptive immune cell interaction drives inflammasome activation and hepatocyte apoptosis in murine liver injury from immune checkpoint inhibitors

Immune checkpoints (CTLA4 & PD-1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune checkpoint inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune-mediated liver injury from ICIs (ILICI). Learning how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance. We developed a murine model to recapitulate human ILICI using CTLA4+/- mice treated with either combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2. We tested two forms of antisense oligonucleotides to knockdown caspase-3 in a total liver (parenchymal and non-parenchymal cells) or in a hepatocyte-specific manner. We also employed imaging mass cytometry (IMC), a powerful multiplex modality for immunophenotyping and cell interaction analysis in our model. ICI-treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as Nod-like receptor protein 3 (NLRP3) inflammasome activation, but no necroptosis. Total liver knockdown of caspase-3 worsened liver injury, and induced further inflammasome activation, and Gasdermin-D-mediated pyroptosis. Hepatocyte-specific knockdown of caspase-3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed that cC3+ hepatocytes had significantly closer interactions with macrophages, Kupffer cells, and NLRP3hi myeloid cells than other cell types. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8 + T-cells, and macrophages. Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Furthermore, we report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.