Immune Surveillance Mechanisms Research Articles

Abstract PO1-13-04: Distinct immune signatures discriminate between patients defined as exceptional survivors of metastatic breast cancer compared to those with rapidly progressing treatment resistant breast cancers

Abstract Background: "Atypical responders" can encompass three sub- categories of patients: "exceptional responders" (those with an unusually favourable treatment response), "rapid progressors" (unusually poor or no response), and "exceptional/long-term survivors" (outlived initial prognosis). Here, we aim to investigate the drivers of immune surveillance mechanisms (local, lymphoid and peripheral) and the nature of immunological tumour recognition in exceptional survivors of stage 4 metastatic disease following standard-of-care chemo- and targeted therapies. Methods: We conducted a complementary multi-platform immune profiling study to define differences in the phenotypic immune landscape including mass-spectrometry-based proteomics on peripheral blood mononuclear cells (PBMCs), multiplex cytokine profiling of patient serum, and high-dimensional flow cytometry on whole blood samples. By staining 500μl of whole blood using 2 panels, 353 immune-cell-related parameters were obtained for comparisons among the following groups: "exceptional survivors," "exceptional responders," "rapid progressors," "non-exceptional metastatic patients," "early breast cancer patients," and "healthy volunteers". Patients were matched for age, breast cancer receptor status, and sites of metastases where possible. Results: Preliminary analysis of serum biomarkers and proteome of the PBMCs of the exceptional survivors showed elevated levels of NK-cell-related proteins such as KCTD10 and RBBP7. Flow cytometry analysis revealed the presence of increased activated CD56dim NK cells, CD56bright NK cells, and central memory (CM) (CD45RA-CD27+) CD8 T cells in both the exceptional survivors and the rapid progressors. However, expression of CD25 appears to be the main mode of activation in rapid progressors compared with increased NKG2D expression in exceptional survivors. Additionally, activation of the unconventional gd T cells was evident in both groups with terminally differentiated effector memory cells re-expressing CD45RA (TEMRA) Vd1 cell predominating in the exceptional survivors. In the rapid progressors, the increased CD25+ TEMRA Vd2 cells coupled with elevated serum IL-17A and IL-1b suggest their ability to generate IL-17. Th2 cytokines, IL-5 and IL-13 were also enriched in rapid progressors and concomitant increase in CD25+ Th2 cells reveals a strong Th2-driven immune signature in these patients. Finally increased CD86+ atypical double negative (DN) B cells, mostly comprised of the DN2 subset were also significantly enriched in the rapid progressors compared to the exceptional survivors where DN1 B cells were prevalent. Conclusion: The multi-platform approach to investigating immune responses present in atypical responders has identified several distinct immunophenotypes in which the extreme outliers differ in their potential immune surveillance mechanisms. Functional validation of these findings through activation and cytotoxicity assays is currently underway and future work aims to understand the spatial biology of these circulating immune cells within the context of their primary tumours, metastases, and lymph nodes. Citation Format: Helen Kakkassery, Thanussuyah Alaguthurai, Zuza Kozik, Paul Buckley, Ruifang Lu, Esme Carpenter, Rosalind Graham, Gheed Alhity, Farhana Hossain, Sadiyah Mukhtar, Syed Haider, Jyoti Choudhary, Sheeba Irshad. Distinct immune signatures discriminate between patients defined as exceptional survivors of metastatic breast cancer compared to those with rapidly progressing treatment resistant breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-04.

Abstract SoA2-02: Implications of therapy induced senescence in breast cancer

Abstract The goal of breast cancer treatment is to cause cellular stress sufficient to kill and eliminate the tumor cells. Unfortunately, in many cases cancer cells survive and are not removed by surgery and thus can cause relapse and eventual mortality. Surviving cells have necessarily avoided both intrinsic (e.g., apoptosis) and extrinsic (e.g., immune surveillance) mechanisms of death. Studies over the last 20 years have shown that chemotherapy-treated breast cancer cells can evade cell death by activating programs of cellular senescence mediated by the tumor suppressor p53. While p53 can induce apoptosis in response to stress in some cell types, in breast cancer, p53 preferentially induces cell cycle arrest and senescence in response to chemotherapy. Data show that patients with breast cancers that are wild type for TP53 have a much lower probability of being eradicated by chemotherapy, and have a much shorter survival, compared to patients with TP53 mutant tumors. Human and mouse mammary tumors induce many markers of senescence after chemotherapy treatment, including those associated with cell cycle arrest and the senescence associated secretory phenotype (SASP). These arrested tumors evade death from mitotic catastrophe and can block apoptotic programs via elevated activity of BCL2 family members. More recent studies have addressed how induction of senescence deters immune surveillance. Breast cancer cells exposed to chemotherapy reorganize chromatin structure that amplifies stromal derived signals that stimulate expression of many immune genes, including checkpoint ligands such as PD-L1 and Galectin-9. Further, p53 can directly transactivate other immune modulatory genes such as CD80. Collectively, these properties of senescent cells enable their persistence following treatment and contribute to their potential to drive relapse. These same properties, however, can also be exploited as vulnerabilities to therapeutics such as senolytic drugs that target reliance on BCL2 family members or immunotherapies that target PD-L1 expression. Citation Format: J. Jackson. Implications of therapy induced senescence in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr SoA2-02.

CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1–CXCR2 induced NETs

BackgroundCD276 (B7-H3), a pivotal immune checkpoint, facilitates tumorigenicity, invasiveness, and metastasis by escaping immune surveillance in a variety of tumors; however, the underlying mechanisms facilitating immune escape in esophageal squamous...

Coagulation Protease-Driven Cancer Immune Evasion: Potential Targets for Cancer Immunotherapy.

Blood coagulation and cancer are intrinsically connected, hypercoagulation-associated thrombotic complications are commonly observed in certain types of cancer, often leading to decreased survival in cancer patients. Apart from the common role in coagulation, coagulation proteases often trigger intracellular signaling in various cancers via the activation of a G protein-coupled receptor superfamily protease: protease-activated receptors (PARs). Although the role of PARs is well-established in the development and progression of certain types of cancer, their impact on cancer immune response is only just emerging. The present review highlights how coagulation protease-driven PAR signaling plays a key role in modulating innate and adaptive immune responses. This is followed by a detailed discussion on the contribution of coagulation protease-induced signaling in cancer immune evasion, thereby supporting the growth and development of certain tumors. A special section of the review demonstrates the role of coagulation proteases, thrombin, factor VIIa, and factor Xa in cancer immune evasion. Targeting coagulation protease-induced signaling might be a potential therapeutic strategy to boost the immune surveillance mechanism of a host fighting against cancer, thereby augmenting the clinical consequences of targeted immunotherapeutic regimens.

Abstract 1369: PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection

Abstract Obesity has been established as a leading risk factor for many cancers and can drive tumor progression and metastasis. Paradoxically, obesity has in some cases been associated with better survival and improved response to immune checkpoint blockade therapies. The role of the immune system in the obesity-cancer connection and how obesity affects immunotherapy responses, however, have been unclear. Here we show that obesity enhanced PD-1 expression on macrophages to reduce phagocytosis and antigen presentation to T cells that correlated with reduced T cell expansion and function. This obesity associated immune dysfunction, however, primed for enhanced anti-tumor response to PD-1 blockade. Single cell RNAseq showed obesity remodels myeloid and T cell populations but does not impact non-immune cell populations such as fibroblasts and epithelial cells. Specifically, obesity increased the overall number of tumor-associated macrophages (TAM) while effector T cells were decreased in abundance. Interestingly, the frequency of macrophages expressing PD-1 increased, while the remaining T cells maintained similar or reduced PD-1 expression and appeared less activated. Further, T cell depletion from the tumor microenvironment, enhanced macrophage PD-1 expression and worsened PD1-associated TAM dysfunction in an obesity enhanced manor. Obesity associated cytokines and adipokines including IFN-ã, leptin and insulin induced PD-1 expression on macrophages. This expression is associated with mTOR and c-myc activation signaling pathways; as such inhibiting these signaling pathways blocked PD1 expression. Bulk RNA sequencing analysis revealed PD-1+ TAMs had an altered gene profile compared to PD-1− TAMs. PD1 expressing macrophages exhibited increased mitochondrial respiration and expression of oxidative phosphorylation, increased lipid uptake and increased cell cycling related genes. Conversely PD1- TAMs exhibited increased phagocytosis and antigen presentation. PD-1/PDL1 interaction directly regulated TAMs, as recombinant PDL-1 reduced glycolysis and phagocytosis in purified macrophages, and these effects could be reversed with blocking PD-1 antibody. Conversely, PD-1−/− TAMs had reduced lipid uptake but high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth and decreased LAG3 and increased CD69 on CD8 T cells. In addition, myeloid specific PD1 deficiency enhanced TAM antigen presentation through OVA specific CD8 T cell activation. These findings identify PD-1 as a metabolic regulator in TAM dysfunction and reveal a unique PD-1 mediated and macrophage-specific mechanism for immune tumor surveillance and checkpoint blockade. This may contribute to improved immunotherapy response in TAM-enriched tumors and obesity. Citation Format: Jackie Bader, Jeffrey Rathmell. PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection [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 1369.

Abstract 486: Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with a 5-year survival rate of less than 13% due to the lack of effective diagnostic/therapeutic modalities. Paclitaxel (PTX) has been tested in pancreatic cancer (PanCa) therapy with marginally better clinical outcomes, but remains limited by its poor hemocompatibility, biodistribution, and intracellular accumulation in tumor cells. Gemcitabine (GEM) is the most effective therapy for PanCa. However, it shows only a marginal survival benefit of 6 months. The poor therapy response is attributed to high desmoplasia, unfavorable tumor microenvironment (TME), and excessive recruitment of immune suppressive cells. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated paclitaxel loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PPNPs), which effectively inhibits pancreatic tumor. In this study, we demonstrate that PPNPs effectively sensitize tumor cells to GEM via attenuation of tumor desmoplasia. Our findings also show that PPNPs synergize GEM therapy response in cell lines and in vivo mouse models. Mechanistically, PPNPs target the TME via inhibition of the sonic hedgehog (SHH) pathway and other oncogenic signaling axis that inhibits bidirectional tumor-stromal cell interaction as determined by qPCR and Western blot analyses. In addition, PPNPs effectively target tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in the presence of PPNPs or vehicle control. Furthermore, PPNPs effectively increase phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that our novel PPNP nano formulation effectively targets TME, and facilitates GEM uptake by inhibiting the activation of SHH signaling and reprograming the tumor immune surveillance mechanisms. This study suggests that PPNPs have great potential for future clinical use in management of pancreatic cancer. Citation Format: Vivek K. Kashyap, Godwin P. Darkwah, Neeraj Chauhan, Mohammed Sikander, Eswara N. Ghali, Meghana Kolli, Bilal B. Hafeez, Murali M. Yallapu, Subhash C. Chauhan. Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses [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 486.

H2-K1 on MLL-AF9 Leukemia Cells Facilitates the Escape of NK Cell-Mediated Immune Surveillance

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, and prognosis is poor; 5-year survival approximately 30%. Recently, there is an emerging recognition of the innate immune system, in particular Natural killer (NK) cells and macrophages, for immune surveillance against AML, but the mechanistic basis for this is mostly unknow. Identifying how AML cells escape immune surveillance by NK cells may translate into new treatment opportunities for AML patients. To identify new therapeutic opportunities, we recently performed an in vivo CRISPR/Cas9 screen targeting 961 cell surface genes using the MLL-AF9 AML mouse model. One of the top leukemia stem cell dependencies in the bone marrow niche was H2-K1, an ortholog of human HLA-A, a classical MHC class-I molecule. In validation experiments, we observed a seven-fold depletion (p<0.001) of H2-K1 sgRNA-expressing c-Kit +Cas9 + leukemia cells in the bone marrow. In contrast, genetic disruption of H2-K1 did not impact the growth and survival of MLL-AF9 leukemia cells in culture. Given the known suppressive role of MHC class-I molecules for immune cells, we speculated that H2-K1 mayprovide inhibitory signals that counteract immune-surveillance mechanisms in the bone marrow niche. To test this hypothesis, we depleted NK cells and macrophages prior to transplantation of the c-Kit + MLL-AF9 leukemia cells. Macrophage depletion by clodronate liposomes did not affect the in vivo expansion of H2-K1 sgRNA-expressing leukemia cells demonstrating that macrophages were not suppressed by H2-K1 on the leukemia cells. In contrast, NK1.1 antibody-mediated depletion of NK cells fully rescued the depletion of H2-K1 sgRNA-expressing leukemia cells in vivo. These findings suggest that H2-K1 expression on MLL-AF9 leukemia cells inhibits NK cells in this model. Consistent with these findings, H2-K1 knockdown in leukemia cells triggered a two-fold increase of INFγ production (p<0.01) in the NK cells, accompanied by augmented apoptosis of the leukemia cells (p<0.01). Given that NK cells have been shown to be dysfunctional in AML patients, we next explored whether leukemia development affects NK cell maturation. The expansion of leukemia cells in the mice skewed NK cells towards a M1 (CD27 +CD11b -) state and decreased the level of the more cytotoxic M2 (CD27 +CD11b +) and M3 (CD27 -CD11b +) populations. H2-K1 disruption in the leukemic cells restored the level of M2 and M3 NK cell population in the bone marrow. Notably, restoration of matured NK cell populations was accompanied by an increased expression of NKG2D, an activating receptor, indicating a more cytotoxic state of the NK cells. In line with these findings, ablation of H2-K1 in leukemia cells induced JAK/STAT and NF-κβ signaling in the NK cells. Taken together, our study identifies that H2-K1 on MLL-AF9 leukemia stem cells facilitates immune evasion by suppressing NK cells. H2-K1 alters the maturation and activation of NK cells in the bone marrow niche. These findings increase our understanding of how leukemia cells escape immune surveillance and suggest that the identification of corresponding mechanisms in human AML could pave the way for new therapies that boost the endogenous NK cells by restoring immune surveillance mechanisms.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p < 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

Vaccine and Adjuvant Mediated Autoimmunity

The macrophage, natural killer cells, B and T cells are the outstanding formed elements of human immune system. In normal immune homeostatic state these cells in a way or other recognize the host body components as self via the immune surveillance mechanisms. Though when there was a shift in immune homeostasis due to  chronic induction by; i - environmental stimulus, ii –interplay of predisposing genetic elements, iii-family history, iv – bystander pathologic inflammatory system .Immune cells becomes prone to recognize the impart self or self as a non-self with subsequent induction of autoimmune diseases. The present opinion paper was aimed at vaccine and adjuvant mediated autoimmune diseases. Timelines for vaccine and adjuvant induced autoimmunity were made. Different human approved vaccines induce different autoimmune disease, more than on vaccine may induce same autoimmune disease. Shoenfeld Syndrome, the adjuvant induce autoimmune/ inflammatory syndrome. Under the umbrella of this syndrome five conditions grouped as; i – Postvaccination with an adjuvanated vaccine, ii – macrophagic myofasciitis, iii – sick building disease condition iv – Gulf war disease condition and v – siliconosis. Protocol for the practical evaluation of these diseases was suggested. Understanding Shoenfeld Syndrome is crucial for producing vaccines with a safer side effect profile.

Distinct immune surveillance in primary biliary cholangitis and primary sclerosing cholangitis is linked with discrete cholangiocarcinoma risk.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are 2 major liver autoimmune diseases. PBC is common in women and primarily affects intrahepatic small bile duct epithelial cells, known as cholangiocytes. In contrast, PSC is dominant in men and primarily affects medium and big intrahepatic and extrahepatic bile duct epithelial cells. Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, and its incidence is increasing worldwide in both men and women. Numerous retrospective and clinical studies have suggested that PBC patients rarely develop CCA compared to PSC patients. CCA is accountable for the higher deaths in PSC patients due to ineffective therapies and our inability to diagnose the disease at an early stage. Therefore, it is paramount to understand the differences in immune surveillance mechanisms that render PBC patients more resistant while PSC patients are susceptible to CCA development. Here, we review several potential mechanisms contributing to differences in the susceptibility to CCA in PBC versus PSC patients.

IMMUNOREACT 6: weak immune surveillance characterizes early-onset rectal cancer.

Colon cancer in young patients is often associated with hereditary syndromes; however, in early-onset rectal cancer, mutations of these genes are rarely observed. The aim of this study was to analyse the features of the local immune microenvironment and the mutational pattern in early-onset rectal cancer. Commonly mutated genes were analysed within a rectal cancer series from the University Hospital of Padova. Mutation frequency and immune gene expression in a cohort from The Cancer Genome Atlas ('TCGA') were compared and immune-cell infiltration levels in the healthy rectal mucosa adjacent to rectal cancers were evaluated in the IMMUNOlogical microenvironment in REctal AdenoCarcinoma Treatment 1 and 2 ('IMMUNOREACT') series. In the authors' series, the mutation frequency of BRAF, KRAS, and NRAS, as well as microsatellite instability frequency, were not different between early- and late-onset rectal cancer. In The Cancer Genome Atlas series, among the genes with the most considerable difference in mutation frequency between young and older patients, seven genes are involved in the immune response and CD69, CD3, and CD8β expression was lower in early-onset rectal cancer. In the IMMUNOlogical microenvironment in REctal AdenoCarcinoma Treatment 1 and 2 series, young patients had a lower rate of CD4+ T cells, but higher T regulator infiltration in the rectal mucosa. Early-onset rectal cancer is rarely associated with common hereditary syndromes. The tumour microenvironment is characterized by a high frequency of mutations impairing the local immune surveillance mechanisms and low expression of immune editing-related genes. A constitutively low number of CD4 T cells associated with a high number of T regulators indicates an imbalance in the immune surveillance mechanisms.

Association of CD47 Expression with Clinicopathologic Characteristics and Survival Outcomes in Muscle Invasive Bladder Cancer.

CD47 is an antiphagocytic molecule that plays a critical role in immune surveillance. A variety of malignancies have been shown to evade the immune system by increasing the expression of CD47 on the cell surface. As a result, anti-CD47 therapy is under clinical investigation for a subset of these tumors. Interestingly, CD47 overexpression is associated with negative clinical outcomes in lung and gastric cancers; however, the expression and functional significance of CD47 in bladder cancer is not fully understood. We retrospectively studied patients with muscle invasion bladder cancer (MIBC) who underwent a transurethral resection of bladder tumor (TURBT) and subsequently underwent radical cystectomy (RC) with or without neoadjuvant chemotherapy (NAC). CD47 expression was examined by IHC in both TURBT and matched RC specimens. The difference in CD47 expression levels between TURBT and RC was also compared. The association of CD47 levels (TURBT) with clinicopathological parameters and survival outcomes was evaluated by Pearson's chi-squared tests and the Kaplan-Meier method, respectively. A total of 87 MIBC patients were included. The median age was 66 (39-84) years. Most patients were Caucasian (95%), male (79%), and aged >60 (63%) and most often (75%) underwent NAC prior to RC. Of those who received NAC, 35.6% were responders and 64.4% were non-responders. The final reported stages as per AJCC for all patients were as follows: stage 0 (32%), stage 1 (1%), stage 2 (20%), stage 3 (43%), and stage 4a (5%). A total of 60% of patients were alive; of those, 30% had disease recurrence and 40% died from bladder cancer at a median follow-up of 3.1 (0.2-14.2) years. CD47 levels were detectable in 38 (44%) TURBT samples. There was no association between CD47 levels and clinicopathological parameters such as age, gender, race, NAC, final stage, disease recurrence, and overall survival (OS). Patients aged >60 (p = 0.006), non-responders (p = 0.002), and at stage ≥ 3 (p < 0.001) were associated with worse OS by a univariate analysis and stage ≥ 3 remained significant even after a multivariate analysis. In patients managed with NAC, there were decreased CD47 levels in RC specimens compared to the TURBT specimens, but this did not reach statistical significance. CD47 expression was not a predictive nor prognostic marker for MIBC patients. However, expression of CD47 was detected in nearly half of MIBCs, and future studies are needed to explore the potential role of anti-CD47 therapy in these patients. Furthermore, there was a slight positive trend in decreased CD47 levels (from TURBT to RC) in patients receiving NAC. As a result, more research is needed to understand how NAC may modify immune surveillance mechanisms in MIBC.

Global research trends in immunotherapy for head and neck neoplasms: A scientometric study

In recent decades, the traditional treatment of head and neck neoplasms has reached a bottleneck with limited improvement in overall survival. Nevertheless, the emerging field of immunotherapy has shown promise. Literature on research into immunotherapy for head and neck neoplasms was retrieved from WoSCC. Citespace was used as a scientometric analysis tool for text mining and visualization of the scientific literature. This analysis included 1915 documents. Recently, the annual number of publications and citations has been growing rapidly. ‘Oncology’ was the most popular research area. The most dominant institution and country were the University of Pittsburgh and the USA. Ferris RL was not only the most prolific but also the most cited author, demonstrating a strong influence and reputation. Of the ten core journals identified in this field, Cancer Research ranked first. ‘Regulatory T cell’, ‘PD-1’ and ‘biomarker’ were regarded as current hotspots, while ‘recurrent’ and ‘nivolumab’ were considered as trending keywords. The most cited reference was Ferris RL (2016). Notably, the front trends and future directions in the field may lie in the clinical practice of combination therapy of immunotherapy plus other therapies, the mechanism of impaired immune surveillance, and the improvement in resistance to immunotherapeutic agents. It is firmly believed that the present scientometric analysis has provided both a macroscopic and microscopic overview of research into immunotherapy for head and neck neoplasms, which will assist researchers and oncologists to better understand this discipline and thus promote further development and policies in this field.

Abstract PD2-01: Local and lymphoid immune surveillance mechanisms in “exceptional survivors” of stage 4 cancers following standard of care chemo- and targeted therapies

Abstract Breast cancer patients with advanced metastatic disease can exhibit rapid disease progression, disease stabilisation or partial responses of varying duration. However, for reasons that are not fully elucidated, a small fraction of patients will elicit an exceptional durable response to standard anticancer treatments or survive significantly longer than patients with clinically comparable tumours. Here, we investigate the drivers of immune surveillance mechanisms across breast cancer subtypes in stage IV exceptional survivors (n=13) with matched control cohorts of stage IV typical responders (n=6), early breast cancer patients (n=5) and healthy volunteers (n=17). Peripheral blood mononuclear cells (PBMCs) from patients were stained with 8 panels providing 241 non-redundant immune parameters for flow cytometry analysis. Principle Component Analysis (PCA) showed distinct segregation of the exceptional survivors from the other control groups with an immune signature in exceptional survivors constituting of activated NK, CD8 T cells and gamma delta (gd) T cells pointing towards higher innate immunogenicity in these individuals. Specifically, although these metastatic exceptional responder patients possessed comparable NK cell frequencies, the proportion of NKG2D+CD56dimCD16+ NK cells were significantly enriched compared to the typical responders. Additionally, proportions of CD8+ central memory (CD45RA- CD27+) and effector memory (CD45RA- CD27-) gd T cells, were also seen to be significantly increased. Functional in vitro validation of these findings along with scRNA sequencing of lymph node and tumour tissue is currently underway. To our knowledge, this work is the first to explore in depth the immune signatures in the peripheral blood of exceptional survivors with metastatic breast cancer. Elucidating the immunological reasons for favourable atypical responses alongside functional tumour microenvironment analysis offers unique insights for predictive biomarker identification and discovery of axes that could be exploited therapeutically to benefit those with less favourable responses. Written informed consent was obtained from all individuals in accordance with the Declaration of Helsinki under the following research ethics committee; London-Chelsea approved study (REC ID 13/LO/1248). Citation Format: Helen Kakkassery, Thanussuyah Alaguthurai, Rosalind Graham, Esme Carpenter, Farhana Hossain, Sean Keane, Sheeba Irshad. Local and lymphoid immune surveillance mechanisms in “exceptional survivors” of stage 4 cancers following standard of care chemo- and targeted therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-01.

Prognostic significance of PD-1, CTLA-4, CD4, and CD8 expression in olfactory neuroblastoma.

There are limited data regarding immune surveillance mechanisms in olfactory neuroblastoma. We investigated the expression of programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD4, and CD8 in olfactory neuroblastoma to identify potential therapeutic targets. Immunohistochemistry was used to detect PD-1 and CTLA-4 and measure the numbers of CD4+ and CD8+ T cells in 56 patients with olfactory neuroblastoma. The relationships between these molecules in tumor microenvironment, clinicopathological features, and survival were analyzed. The prevalence of PD-1 in Kadish C stage was 24.14%, significantly greater than in Kadish A and B stage. CD4+ T-cell and CD8+ T-cell levels correlated with higher Hyams histological grade and Kadish stage. In addition, PD-1 was related positively with CTLA-4, CD4+ T cells, and CD8+ T cells in olfactory neuroblastoma. Univariate survival analysis showed that higher PD-1 positivity, CD8+ T cells, and Hyams grade correlated with worse clinical outcome. Multivariate analysis showed that the expression of PD-1 was an independent parameter for poor prognosis. In conclusion, olfactory neuroblastoma with PD-1 expression had more aggressive clinicopathological features and worse prognosis. PD-1 may potentially predict the outcome of olfactory neuroblastoma patients.

Review on host-pathogen interaction in dermatophyte infections.

Dermatophytosis is a common superficial fungal infection of the skin and its appendages caused by dermatophytes. Recent times have witnessed a dynamic evolution of dermatophytes driven by their ecology, reproduction, pathogenicity and host immune response, influenced by population migration and socioeconomic status. Dermatophytes establish infection following successful adherence of arthroconidia to the surface of keratinized tissues. The proteolytic enzymes released during adherence and invasion not only ascertain their survival but also allow the persistence of infection in the host. While the cutaneous immune surveillance mechanism, after antigen exposure and presentation, leads to activation of T lymphocytes and subsequent clonal expansion generating effector T cells that differentially polarize to a predominant Th17 response, the response fails to eliminate the pathogen despite the presence of high levels of IFN-γ. In chronic dermatophytosis, antigens are a constant source of stimulus promoting a dysregulated Th17 response causing inflammation. The host-derived iTreg response fails to counterbalance the inflammation and instead polarizes to Th17 lineage, aggravating the chronicity of the infection. Increasing antifungal resistance and recalcitrant dermatophytosis has impeded the overall clinical remission. Human genetic research has the potential to generate knowledge to explore new therapeutic targets. The review focuses on understanding specific virulence factors involved in pathogenesis and defining the role of dysregulated host immune response against chronic dermatophytic infections for future management strategies.

Abstract A007: Assessing immunoprevention efficacy of an agonist of CD137 pathway in a tobacco carcinogen-induced lung cancer preclinical model

Abstract Purpose: Lung cancer is the leading cause of cancer-related deaths worldwide. Cigarette smoking is the primary risk factor for lung cancer and is linked to 80-90% of lung cancer deaths according to the Centers for Disease Control (CDC). The most carcinogenic component of tobacco smoke is NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). SA-4-1BBL is a novel CD137 receptor agonist that has shown cancer immunoprevention efficacy in transplantable preclinical tumor models by invoking an innate immune surveillance mechanism involving CD4+ CD44+ T and NK cells. As a preclinical model for high-risk populations, we evaluated the immunoprevention efficacy of SA-4-1BBL in lung tumor formation induced by NNK. Experimental Procedures: A/J female mice were used as a preclinical model for lung cancer immunoprevention due to their susceptibility to tobacco-carcinogen-induced lung cancer. A/J mice were i.p. injected with NNK weekly for eight consecutive treatments. The mice were also treated s.c. with 100 µg SA-4-1BBL twice, two weeks apart on weeks 6 and 8 of NNK treatment. Animals were monitored for 21 weeks and euthanized to harvest the lung and lung-draining lymph nodes for histology (H&amp;E staining and PCNA immunofluorescence staining). In separate groups, mice were treated with a depleting antibody to CD4 twice two weeks apart on weeks 6 and 8 of NNK treatment to investigate the role of CD4+ immune cells in cancer immunoprevention. Results: NNK treatment resulted in lung tumor formation as assessed by PCNA immunofluorescence staining in all saline-injected control mice by the 21-week experimental end-point. Treatment with SA-4-1BBL resulted in significantly fewer macroscopic and microscopic tumor numbers than the control group (p &amp;lt; 0.01). Animals treated with SA-4-1BBL and subjected to CD4+ cell depletion showed similar numbers of macroscopic and microscopic lung tumors to the vehicle group. Conclusions: Prophylactic treatment with SA-4-1BBL significantly prevents NNK-induced lung tumor formation and CD4+ immune cells play a critical role in the observed immunoprevention efficacy of SA-4-1BBL. Supported by a grant from DOD LC190524 to Esma S. Yolcu. Citation Format: Ayse Ece Gulen, Mohammad Tarique, Vahap Ulker, Evrim Agca, Haval Shirwan, Esma S. Yolcu. Assessing immunoprevention efficacy of an agonist of CD137 pathway in a tobacco carcinogen-induced lung cancer preclinical model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A007.

Anti-tumour potential of PD-L1/PD-1 post-translational modifications.

The immune checkpoint programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) are biologically important immunosuppressive molecules, and the PD-L1/PD-1-mediated signalling pathway is currently considered one of the main mechanisms of tumour escape immune surveillance. PD-L1 is highly expressed on the cytomembrane of tumour cell and binds to PD-1 receptor of activated T cells. This interaction activates PD-L1/PD-1 downstream signal transduction, inhibiting T cells anti-tumour activity. Therefore, inhibitors of PD-L1/PD-1 activation, showing significant efficacy in some types of tumours, have been widely approved in clinical tumour therapy. Recent research on PD-L1/PD-1 signalling pathway regulation has shown post-translational modifications (PTMs) form of PD-L1 or PD-1, including glycosylation, ubiquitination, phosphorylation, and acetylation, which may play an important role in PD-L1/PD-1 signalling pathway regulation and anti-tumour function of T cells. In this review, we focused on PTMs of PD-L1/PD-1 research and potential applications in tumour immunotherapy.

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

BackgroundMetastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC.MethodsA cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita’s overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher’s, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method.ResultsStratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03).ConclusionThese data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.

Genomic crossroads between non-Hodgkin's lymphoma and common variable immunodeficiency.

Common variable immunodeficiency (CVID) represents the largest group of primary immunodeficiencies that may manifest with infections, inflammation, autoimmunity, and cancer, mainly B-cell non-Hodgkin’s lymphoma (NHL). Indeed, NHL may result from chronic or recurrent infections and has, therefore, been recognized as a clinical phenotype of CVID, although rare. The more one delves into the mechanisms involved in CVID and cancer, the stronger the idea that both pathologies can be a reflection of the same primer events observed from different angles. The potential effects of germline variants on specific somatic modifications in malignancies suggest that it might be possible to anticipate critical events during tumor development. In the same way, a somatic alteration in NHL could be conditioning a similar response at the transcriptional level in the shared signaling pathways with genetic germline alterations in CVID. We aimed to explore the genomic substrate shared between these entities to better characterize the CVID phenotype immunodeficiency in NHL. By means of an in-silico approach, we interrogated the large, publicly available datasets contained in cBioPortal for the presence of genes associated with genetic pathogenic variants in a panel of 50 genes recurrently altered in CVID and previously described as causative or disease-modifying. We found that 323 (25%) of the 1,309 NHL samples available for analysis harbored variants of the CVID spectrum, with the most recurrent alteration presented in NHL occurring in PIK3CD (6%) and STAT3 (4%). Pathway analysis of common gene alterations showed enrichment in inflammatory, immune surveillance, and defective DNA repair mechanisms similar to those affected in CVID, with PIK3R1 appearing as a central node in the protein interaction network. The co-occurrence of gene alterations was a frequent phenomenon. This study represents an attempt to identify common genomic grounds between CVID and NHL. Further prospective studies are required to better know the role of genetic variants associated with CVID and their reflection on the somatic pathogenic variants responsible for cancer, as well as to characterize the CVID-like phenotype in NHL, with the potential to influence early CVID detection and therapeutic management.