LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

Perivascular tumor-associated macrophages and their role in cancer progression.

Perivascular (Pv) tumor-associated macrophages (TAMs) are a highly specialized stromal subset within the tumor microenvironment (TME) that are defined by their spatial proximity, within one cell thickness, to blood vasculature. PvTAMs have been demonstrated to support a variety of pro-tumoral functions including angiogenesis, metastasis, and modulating the immune and stromal landscape. Furthermore, PvTAMs can also limit the response of anti-cancer and anti-angiogenic therapies and support tumor recurrence post-treatment. However, their role may not exclusively be pro-tumoral as PvTAMs can also have immune-stimulatory capabilities. PvTAMs are derived from a monocyte progenitor that develop and localize to the Pv niche as part of a multistep process which relies on a series of signals from tumor, endothelial and Pv mesenchymal cell populations. These cellular communications and signals create a highly specialized TAM subset that can also form CCR5-dependent multicellular 'nest' structures in the Pv niche. This review considers our current understanding of the role of PvTAMs, their markers for identification, development, and function in cancer. The role of PvTAMs in supporting disease progression and modulating the outcome from anti-cancer therapies highlight these cells as a therapeutic target. However, their resistance to pan-TAM targeting therapies, such as those targeting the colony stimulating factor-1 (CSF1)-CSF1 receptor axis, prompts the need for more targeted therapeutic approaches to be considered for this subset. This review highlights potential therapeutic strategies to target and modulate PvTAM development and function in the TME.

Antibody conjugates for targeted delivery of Toll-like receptor 9 agonist to the tumor tissue.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.

Tinker, tailor, soldier, cell: the role of C-type lectins in the defense and promotion of disease.

C-type lectins (CTLs) represent a large family of soluble and membrane-bound proteins which bind calcium dependently via carbohydrate recognition domains (CRDs) to glycan residues presented on the surface of a variety of pathogens. The deconvolution of a cell's glycan code by CTLs underpins several important physiological processes in mammals such as pathogen neutralization and opsonization, leukocyte trafficking, and the inflammatory response. However, as our knowledge of CTLs has developed it has become apparent that the role of this innate immune family of proteins can be double-edged, where some pathogens have developed approaches to subvert and exploit CTL interactions to promote infection and sustain the pathological state. Equally, CTL interactions with host glycoproteins can contribute to inflammatory diseases such as arthritis and cancer whereby, in certain contexts, they exacerbate inflammation and drive malignant progression. This review discusses the 'dual agent' roles of some of the major mammalian CTLs in both resolving and promoting infection, inflammation and inflammatory disease and highlights opportunities and emerging approaches for their therapeutic modulation.

Predicting progression-free survival after systemic therapy in advanced head and neck cancer: Bayesian regression and model development.

Background: Advanced head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis, and biomarkers that predict response to treatment are highly desirable. The primary aim was to predict progression-free survival (PFS) with a multivariate risk prediction model. Methods: Experimental covariates were derived from blood samples of 56 HNSCC patients which were prospectively obtained within a Phase 2 clinical trial (NCT02633800) at baseline and after the first treatment cycle of combined platinum-based chemotherapy with cetuximab treatment. Clinical and experimental covariates were selected by Bayesian multivariate regression to form risk scores to predict PFS. Results: A 'baseline' and a 'combined' risk prediction model were generated, each of which featuring clinical and experimental covariates. The baseline risk signature has three covariates and was strongly driven by baseline percentage of CD33+CD14+HLADRhigh monocytes. The combined signature has six covariates, also featuring baseline CD33+CD14+HLADRhigh monocytes but is strongly driven by on-treatment relative change of CD8+ central memory T cells percentages. The combined model has a higher predictive power than the baseline model and was successfully validated to predict therapeutic response in an independent cohort of nine patients from an additional Phase 2 trial (NCT03494322) assessing the addition of avelumab to cetuximab treatment in HNSCC. We identified tissue counterparts for the immune cells driving the models, using imaging mass cytometry, that specifically colocalized at the tissue level and correlated with outcome. Conclusions: This immune-based combined multimodality signature, obtained through longitudinal peripheral blood monitoring and validated in an independent cohort, presents a novel means of predicting response early on during the treatment course. Funding: Daiichi Sankyo Inc, Cancer Research UK, EU IMI2 IMMUCAN, UK Medical Research Council, European Research Council (335326), Merck Serono. Cancer Research Institute, National Institute for Health Research, Guy's and St Thomas' NHS Foundation Trust and The Institute of Cancer Research. Clinical trial number: NCT02633800.

The effects of radiation therapy on the macrophage response in cancer.

The efficacy of radiotherapy, a mainstay of cancer treatment, is strongly influenced by both cellular and non-cellular features of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are a heterogeneous population within the TME and their prevalence significantly correlates with patient prognosis in a range of cancers. Macrophages display intrinsic radio-resistance and radiotherapy can influence TAM recruitment and phenotype. However, whether radiotherapy alone can effectively "reprogram" TAMs to display anti-tumor phenotypes appears conflicting. Here, we discuss the effect of radiation on macrophage recruitment and plasticity in cancer, while emphasizing the role of specific TME components which may compromise the tumor response to radiation and influence macrophage function. In particular, this review will focus on soluble factors (cytokines, chemokines and components of the complement system) as well as physical changes to the TME. Since the macrophage response has the potential to influence radiotherapy outcomes this population may represent a drug target for improving treatment. An enhanced understanding of components of the TME impacting radiation-induced TAM recruitment and function may help consider the scope for future therapeutic avenues to target this plastic and pervasive population.

Immune cell-antibody interactions in health and disease.

The human immune system safeguards against pathogens through a multitude of cellular and molecular signals, involving different components of the innate and adaptive response. Contrastingly, autoimmune diseases, allergic conditions, and cancer evoke different aspects of these otherwise protective processes. Understanding the immunological hallmarks for each pathological setting is essential for improving prevention, diagnosis, prognosis, and treatment. The activatory states of immune effector cells, especially in relation to their direct or indirect interactions with antibodies, are important determinants of an efficient, protective response that results in target clearance and improved clinical outcomes. Dysregulation of effector cells and their functions alongside alternatively activated humoral immune responses may contribute to several chronic diseases including allergic inflammation, autoimmune disorders and cancer. This Review Series brings to the forefront several key activation and regulatory features of immune effector cells in different diseases including cancer, infection allergy, and autoimmunity. Specific attention is drawn on how antibodies can impact effector cell states, and their pro-inflammatory and immune protective functions. Articles in this Series discuss different effector cells and antibody isotypes in infection, inflammation, tolerance and cancer immune surveillance, covering basic and translational mechanisms, clinical and epidemiological insights into these immune responses. Understanding the critical attributes of immune cells, especially those needed to effectively engage antibodies, will undoubtedly help better exploit their potential for disease management and therapy.

Macrophages in ovarian cancer and their interactions with monoclonal antibody therapies.

The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs), which have largely failed to overcome tumour-associated immunosuppression, restrict cancer growth, and significantly improve survival. In recent years, experimental mAb design has moved away from solely targeting ovarian tumours and instead sought to modulate the wider tumour microenvironment (TME). Tumour-associated macrophages (TAMs) may represent an attractive therapeutic target for mAbs in ovarian cancer due to their high abundance and close proximity to tumour cells and their active involvement in facilitating several pro-tumoural processes. Moreover, the expression of several antibody crystallisable fragment (Fc) receptors and broad phenotypic plasticity of TAMs provide opportunities to modulate TAM polarisation using mAbs to promote anti-tumoural phenotypes. In this review, we discuss the role of TAMs in ovarian cancer TME and the emerging strategies to target the contributions of these cells in tumour progression through the rationale design of mAbs.

Macrophages orchestrate the expansion of a proangiogenic perivascular niche during cancer progression.

Tumor-associated macrophages (TAMs) are a highly plastic stromal cell type that support cancer progression. Using single-cell RNA sequencing of TAMs from a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we characterize a subset of these cells expressing lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve-1) that spatially reside proximal to blood vasculature. We demonstrate that Lyve-1+ TAMs support tumor growth and identify a pivotal role for these cells in maintaining a population of perivascular mesenchymal cells that express α-smooth muscle actin and phenotypically resemble pericytes. Using photolabeling techniques, we show that mesenchymal cells maintain their prevalence in the growing tumor through proliferation and uncover a role for Lyve-1+ TAMs in orchestrating a selective platelet-derived growth factor­CC­dependent expansion of the perivascular mesenchymal population, creating a proangiogenic niche. This study highlights the inter-reliance of the immune and nonimmune stromal network that supports cancer progression and provides therapeutic opportunities for tackling the disease.

Generation of hypoxia-sensing chimeric antigen receptor T cells.

Exploiting hypoxia in solid malignancies to restrict expression of chimeric antigen receptors (CARs) on engineered T cells to the tumor microenvironment overcomes the risk of on-target off-tumor toxicity and minimizes tonic signaling, which promotes CAR T cell exhaustion. This protocol summarizes the synthetic biology underlying the development of a stringent oxygen-sensitive CAR for in vitro and in vivo preclinical characterization. For complete details on the use and execution of this protocol, please refer to Kosti et al. (2021).

Hypoxia-sensing CAR T cells provide safety and efficacy in treating solid tumors.

Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies.

The Diverse Roles of Heme Oxygenase-1 in Tumor Progression.

Heme oxygenase-1 (HO-1) is an inducible intracellular enzyme that is expressed in response to a variety of stimuli to degrade heme, which generates the biologically active catabolites carbon monoxide (CO), biliverdin and ferrous iron (Fe2+). HO-1 is expressed across a range of cancers and has been demonstrated to promote tumor progression through a variety of mechanisms. HO-1 can be expressed in a variety of cells within the tumor microenvironment (TME), including both the malignant tumor cells as well as stromal cell populations such as macrophages, dendritic cells and regulatory T-cells. Intrinsically to the cell, HO-1 activity provides antioxidant, anti-apoptotic and cytoprotective effects via its catabolites as well as clearing toxic intracellular heme. However, the catabolites of heme degradation can also diffuse outside of the cell to extrinsically modulate the wider TME, influencing cellular functionality and biological processes which promote tumor progression, such as facilitating angiogenesis and metastasis, as well as promoting anti-inflammation and immune suppression. Pharmacological inhibition of HO-1 has been demonstrated to be a promising therapeutic approach to promote anti-tumor immune responses and inhibit metastasis. However, these biological functions might be context, TME and cell type-dependent as there is also conflicting reports for HO-1 activity facilitating anti-tumoral processes. This review will consider our current understanding of the role of HO-1 in cancer progression and as a therapeutic target in cancer.

ImmunoCluster provides a computational framework for the nonspecialist to profile high-dimensional cytometry data.

High-dimensional cytometry is an innovative tool for immune monitoring in health and disease, and it has provided novel insight into the underlying biology as well as biomarkers for a variety of diseases. However, the analysis of large multiparametric datasets usually requires specialist computational knowledge. Here, we describe ImmunoCluster (https://github.com/kordastilab/ImmunoCluster), an R package for immune profiling cellular heterogeneity in high-dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a nonspecialist. The analysis framework implemented within ImmunoCluster is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users' needs. The protocol consists of three core computational stages: (1) data import and quality control; (2) dimensionality reduction and unsupervised clustering; and (3) annotation and differential testing, all contained within an R-based open-source framework.

Cytotoxic Chemotherapy as an Immune Stimulus: A Molecular Perspective on Turning Up the Immunological Heat on Cancer.

Cytotoxic chemotherapeutics (CCTs) are widely used in the treatment of cancer. Although their mechanisms of action have been best understood in terms of targeting the apparatus of mitosis, an ability to stimulate anti-tumor immune responses is increasing the recognition of these agents as immunotherapies. Immune checkpoint blockade antibodies neutralize important, but specific, immune-regulatory interactions such as PD-1/PD-L1 and CTLA-4 to improve the anti-tumor immune response. However, CCTs can provide a broad-acting immune-stimulus against cancer, promoting both T-cell priming and recruitment to the tumor, which compliments the effects of immune checkpoint blockade. A key pathway in this process is "immunogenic cell death" (ICD) which occurs as a result of tumor cell endoplasmic reticulum stress and apoptosis elicited by CCTs. ICD involves a series of non-redundant signaling events which break tolerance and license anti-tumor antigen-specific T-cells, allowing CCTs to act as "in situ" tumor vaccination tools. Not all responses are tumor cell-intrinsic, as CCTs can also modulate the broader tumor microenvironment. This modulation occurs through preferential depletion of stromal cells which suppress and neutralize robust anti-tumor immune responses, such as myeloid cell populations and Tregs, while effector CD8+ and CD4+ T-cells and NK cells are relatively spared. The immune-stimulating effects of CCTs are dependent on chemotherapy class, dose and tumor cell sensitivity to the agent, highlighting the need to understand the underlying biology of these responses. This mini review considers the immune-stimulating effects of CCTs from a molecular perspective, specifically highlighting considerations for their utilization in the context of combinations with immunotherapy.

Macrophages are exploited from an innate wound healing response to facilitate cancer metastasis.

Tumour-associated macrophages (TAMs) play an important role in tumour progression, which is facilitated by their ability to respond to environmental cues. Here we report, using murine models of breast cancer, that TAMs expressing fibroblast activation protein alpha (FAP) and haem oxygenase-1 (HO-1), which are also found in human breast cancer, represent a macrophage phenotype similar to that observed during the wound healing response. Importantly, the expression of a wound-like cytokine response within the tumour is clinically associated with poor prognosis in a variety of cancers. We show that co-expression of FAP and HO-1 in macrophages results from an innate early regenerative response driven by IL-6, which both directly regulates HO-1 expression and licenses FAP expression in a skin-like collagen-rich environment. We show that tumours can exploit this response to facilitate transendothelial migration and metastatic spread of the disease, which can be pharmacologically targeted using a clinically relevant HO-1 inhibitor.

Perspectives on Chimeric Antigen Receptor T-Cell Immunotherapy for Solid Tumors.

Chimeric antigen receptor (CAR) T-cell therapy entails the genetic engineering of a patient's T-cells to express membrane spanning fusion receptors with defined specificities for tumor-associated antigens. These CARs are capable of eliciting robust T-cell activation to initiate killing of the target tumor cells. This therapeutic approach has produced unprecedented clinical outcomes in the treatment of "liquid" hematologic cancers, but to date has not produced comparable responses in targeting solid malignancies. Advances in our understanding of the immunobiology of solid tumors have highlighted several hurdles which currently hinder the efficacy of this therapy. These barriers include the insufficient accumulation of CAR T-cells in the tumor due to poor trafficking or physical exclusion and the exposure of infiltrating CAR T-cells to a panoply of immune suppressive checkpoint molecules, cytokines, and metabolic stresses that are not conducive to efficient immune reactions and can thereby render these cells anergic, exhausted, or apoptotic. This mini-review summarizes these hurdles and describes some recent approaches and innovations to genetically re-engineer CAR T-cells to counter inhibitory influences found in the tumor microenvironment. Novel immunotherapy drug combinations to potentiate the activity of CAR T-cells are also discussed. As our understanding of the immune landscape of tumors improves and our repertoire of immunotherapeutic drugs expands, it is envisaged that the efficacy of CAR T-cells against solid tumors might be potentiated using combination therapies, which it is hoped may lead to meaningful improvements in clinical outcome for patients with refractory solid malignancies.

Repurposing Tin Mesoporphyrin as an Immune Checkpoint Inhibitor Shows Therapeutic Efficacy in Preclinical Models of Cancer.

Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer.Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations.Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models.Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617-28. ©2018 AACR.

Tumoral immune suppression by macrophages expressing fibroblast activation protein-α and heme oxygenase-1.

The depletion of tumor stromal cells that are marked by their expression of the membrane protein fibroblast activation protein-α (FAP) overcomes immune suppression and allows an anticancer cell immune response to control tumor growth. In subcutaneous tumors established with immunogenic Lewis lung carcinoma cells expressing ovalbumin (LL2/OVA), the FAP(+) population is comprised of CD45(+) and CD45(-) cells. In the present study, we further characterize the tumoral FAP(+)/CD45(+) population as a minor subpopulation of F4/80(hi)/CCR2(+)/CD206(+) M2 macrophages. Using bone marrow chimeric mice in which the primate diphtheria toxin receptor is restricted either to the FAP(+)/CD45(+) or to the FAP(+)/CD45(-) subset, we demonstrate by conditionally depleting each subset that both independently contribute to the immune-suppressive tumor microenvironment. A basis for the function of the FAP(+)/CD45(+) subset is shown to be the immune inhibitory enzyme, heme oxygenase-1 (HO-1). The FAP(+)/CD45(+) cells are the major tumoral source of HO-1, and an inhibitor of HO-1, Sn mesoporphyrin, causes the same extent of immune-dependent arrest of LL2/OVA tumor growth as does the depletion of these cells. Because this observation of immune suppression by HO-1 expressed by the FAP(+)/CD45(+) stromal cell is replicated in a transplanted model of pancreatic ductal adenocarcinoma, we conclude that pharmacologically targeting this enzyme may improve cancer immunotherapy.

Purification, quantification, and functional analysis of Complement Factor H.

Complement Factor H (FH) is an abundant, non-enzymic plasma/serum glycoprotein, which has a major role in regulating activation of the complement system. It can be purified from human plasma/serum by affinity chromatography, using a monoclonal anti-FH antibody as ligand. Other affinity chromatography ligands, including cardiolipin and trinitrophenyl-bovine serum albumin (TNP-BSA), can be used to purify human FH and also FH from a wide range of vertebrates, including mammals, birds, bony fish. Human FH protein concentration can be quantified by sandwich ELISA. The activity of FH is generally measured by assays which detect the cleavage, by complement factor I, of the complement protein C3b to form iC3b. Cleavage occurs only in the presence of a cofactor, and FH is one of a small number of cofactors for this reaction.

Novel glycan biomarkers for the detection of lung cancer.

Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.