Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling.

BACKGROUND: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. METHODS: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. RESULTS: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. CONCLUSIONS: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Warning Signs From the Crypt: Aberrant Protein Glycosylation Marks Opportunities for Early Colorectal Cancer Detection.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths despite being the most preventable and treatable forms of cancer when caught early through screening. There is an unmet need for novel screening approaches with improved accuracy, less invasiveness, and reduced costs. In recent years, evidence has accumulated around particular biological events that happen during the adenoma-to-carcinoma transition, especially focusing on precancerous immune responses in the colonic crypt. Protein glycosylation plays a central role in driving those responses, and recently, numerous reports have been published on how aberrant protein glycosylation both in colonic tissue and on circulating glycoproteins reflects these precancerous developments. The complex field of glycosylation, which exceeds complexity of proteins by several orders of magnitude, can now be studied primarily because of the availability of new high-throughput technologies such as mass spectrometry and artificial intelligence-powered data processing. This has now opened new avenues for studying novel biomarkers for CRC screening. This review summarizes the early events taking place from the normal colon mucosa toward adenoma and adenocarcinoma formation and associated critical protein glycosylation phenomena, both on the tissue level and in the circulation. These insights will help establish an understanding in the interpretation of novel CRC detection modalities that involve high-throughput glycomics.

N-glycolylated carbohydrates in nature.

N-glycolylated carbohydrates are amino sugars with an N-glycolyl amide group. These glycans have not been well studied due to their surprising rarity in nature in comparison with N-acetylated carbohydrates. Recently, however, there has been increasing interest in N-glycolylated sugars because the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), apparently the only source of all N-glycolylated sugars in deuterostomes, appears to be involved in xenosialitis (inflammation associated with consumption of Neu5Gc-rich red meats). Xenosialitis has been implicated in cancers as well as other diseases including atherosclerosis. Furthermore, metabolites of Neu5Gc have been shown to be incorporated into glycosaminoglycans (GAGs), resulting in N-glycolylated GAGs. These N-glycolylated GAGs have important potential applications, such as dating the loss of the Neu5Gc-generating CMAH gene in humans and being explored as a xenosialitis biomarker and/or estimate of the body burden of diet-derived Neu5Gc, to understand the risks associated with the consumption of red meats. This review explores N-glycolylated carbohydrates, how they are metabolized to N-glycolylglucosamine and N-glycolylgalactosamine, and how these metabolites can be incorporated into N-glycolylated GAGs in human tissues. We also discuss other sources of N-glycolylated sugars, such as recombinant production from microorganisms using metabolic engineering as well as chemical synthesis.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

Utilizing Publicly Available Cancer Clinicogenomic Data on CBioPortal to Compare Epidermal Growth Factor Receptor Mutant and Wildtype Non-Small Cell Lung Cancer.

Publicly available clinicogenomic data on platforms such as the cancer BioPortal (cBioPortal.org) allow for efficient analyses by researchers with little or no experience working with Big Data. cBioPortal.org also allows for appropriate statistical testing and downloadable images for easy dissemination of findings. In this study, the cBioPortal.org platform was tested and its utility demonstrated by comparing cases of non-small cell lung cancer (NSCLC) with and without epidermal growth factor receptor gene (EGFR) mutations. Patients with EGFR mutations were more likely to be female, of Asian ethnicity, never-smokers, and be diagnosed with lung adenocarcinoma. Metastasis to the pleura, pleural fluid, and liver was common in patients with EGFR mutant NSCLC. On the other hand, lymph node, brain, and adrenal gland metastases were more common in patients with other mutations. While the median overall survival was about the same in the two groups, progression-free survival was significantly shorter in the EGFR mutant group. The mutational landscape was significantly different in the two groups with EGFR mutant NSCLCs having a lower mutational burden. Differences in copy number alterations between the two groups were also noted. The descriptive data generated from this study such as age, gender, smoking history, and histological subtype recapitulate findings of other studies on EGFR mutant NSCLCs. Further prospective and/or preclinical studies are needed to confirm differences noted in this study. cBioPortal.com queries may be used to supplement clinical/pre-clinical studies or to generate novel hypotheses.

Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis.

Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.

HIV Skews a Balanced Mtb-Specific Th17 Response in Latent Tuberculosis Subjects to a Pro-inflammatory Profile Independent of Viral Load.

HIV infection predisposes latent tuberculosis-infected (LTBI) subjects to active TB. This study is designed to determine whether HIV infection of LTBI subjects compromises the balanced Mycobacterium tuberculosis (Mtb)-specific T helper 17 (Th17) response of recognized importance in anti-TB immunity. Comparative analysis of Mtb- and cytomegalovirus (CMV)-specific CD4+ T cell responses demonstrates a marked dampening of the Mtb-specific CD4+ T cell effectors and polyfunctional cells while preserving CMV-specific response. Additionally, HIV skews the Mtb-specific Th17 response in chronic HIV-infected LTBI progressors, but not long-term non-progressors (LTNPs), with preservation of pro-inflammatory interferon (IFN)-γ+/interleukin-17+ (IL-17+) and significant loss of anti-inflammatory IL-10+/IL-17+ effectors that is restored by anti-retroviral therapy (ART). HIV-driven impairment of Mtb-specific response cannot be attributed to preferential infection as cell-associated HIV DNA and HIV RNA reveal equivalent viral burden in CD4+ T cells from different antigen specificities. We therefore propose that beyond HIV-induced loss of Mtb-specific CD4+ T cells, the associated dysregulation of Mtb-specific T cell homeostasis can potentially enhance the onset of TB in LTBI subjects.

High frequency of exon 20 S768I EGFR mutation detected in malignant pleural effusions: A poor prognosticator of NSCLC.

BACKGROUND: Lung cancer is the cause of a fourth of all cancer-related deaths. About a third of all lung adenocarcinoma tumours harbour mutations on exons 18 to 21 of the epidermal growth factor receptor (EGFR) gene. Detection of these mutations allows for targeted therapies in the form of EGFR Tyrosine kinase inhibitors. Recently, "liquid biopsies" have emerged as an alternative to conventional tissue mutation detection. AIM: In this pilot study, we attempted to optimize EGFR mutation detection from malignant pleural effusions (MPEs) as "liquid biopsies" when tissue biopsies were unavailable. Resulting mutations were then to be mapped on the EGFR gene and explored using cBioPortal, a public cancer genomic database. METHODS AND RESULTS: We first attempted a direct sequencing approach and showed that single nucleotide variants (SNVs) were likely to be missed in MPEs. We then switched to and optimized an EGFR mutant-specific quantitative polymerase chain reaction-based assay. This assay was piloted on n = 10 pleural effusion samples (one non-malignant pleural effusion as a negative control). 5/9 (55.55%) samples harboured EGFR mutations with 2/9 (22.22%) being exon 19 deletions and 3/9 (33.33%) the S768I mutation. The frequency of the S768I SNV in our study was significantly higher than that observed in other studies (~0.2%). Utilizing cBioPortal data, we report that patients with S768I have a shorter median survival time (6 months vs 38 months), progression-free survival time (8 months vs 44 months) and lower tumor mutation count compared to patients with other EGFR mutations. CONCLUSIONS: The shorter survival of patients with the S768I SNV predicts aggressive disease and poor prognosis as a result of this mutation. Studies in larger cohorts and/or animal models are necessary to confirm these findings.

Human species-specific loss of CMP-N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms.

Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD-risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr-/- mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr-/- mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a "xeno-autoantigen" via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc "xeno-autoantibodies" potentiate chronic inflammation ("xenosialitis"). Cmah-/-Ldlr-/- mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah-/-Ldlr-/- mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

From "Serum Sickness" to "Xenosialitis": Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc.

The description of "serum sickness" more than a century ago in humans transfused with animal sera eventually led to identification of a class of human antibodies directed against glycans terminating in the common mammalian sialic acid N-Glycolylneuraminic acid (Neu5Gc), hereafter called "Neu5Gc-glycans." The detection of such glycans in malignant and fetal human tissues initially raised the possibility that it was an oncofetal antigen. However, "serum sickness" antibodies were also noted in various human disease states. These findings spurred further research on Neu5Gc, and the discovery that it is not synthesized in the human body due to a human-lineage specific genetic mutation in the enzyme CMAH. However, with more sensitive techniques Neu5Gc-glycans were detected in smaller quantities on certain human cell types, particularly epithelia and endothelia. The likely explanation is metabolic incorporation of Neu5Gc from dietary sources, especially red meat of mammalian origin. This incorporated Neu5Gc on glycans appears to be the first example of a "xeno-autoantigen," against which varying levels of "xeno-autoantibodies" are present in all humans. The resulting chronic inflammation or "xenosialitis" may have important implications in human health and disease, especially in conditions known to be aggravated by consumption of red meat. In this review, we will cover the early history of the discovery of "serum sickness" antibodies, the subsequent recognition that they were partly directed against Neu5Gc-glycans, the discovery of the genetic defect eliminating Neu5Gc production in humans, and the later recognition that this was not an oncofetal antigen but the first example of a "xeno-autoantigen." Further, we will present comments about implications for disease risks associated with red meat consumption such as cancer and atherosclerosis. We will also mention the potential utility of these anti-Neu5Gc-glycan antibodies in cancer immunotherapy and provide some suggestions and perspectives for the future. Other reviews in this special issue cover many other aspects of this unusual pathological process, for which there appears to be no other described precedent.

Circulating HLA-DR+CD4+ effector memory T cells resistant to CCR5 and PD-L1 mediated suppression compromise regulatory T cell function in tuberculosis.

Chronic T cell activation is a hallmark of pulmonary tuberculosis (PTB). The mechanisms underpinning this important phenomenon are however, poorly elucidated, though known to rely on control of T effector cells (Teff) by regulatory T cells (Treg). Our studies show that circulating natural Treg cells in adults with PTB preserve their suppressive potential but Teff cells from such subjects are resistant to Treg-mediated suppression. We found this to be due to expansion of an activated Teff subset identified by Human Leukocyte Antigen (HLA)-DR expression. Sensitivity to suppression was restored to control levels by depletion of this subset. Comparative transcriptome analysis of Teff cells that contain HLA-DR+ cells versus the fraction depleted of this population identified putative resistance mechanisms linked to IFNG, IL17A, IL22, PD-L1 and ß-chemokines CCL3L3, CCL4 expression. Antibody blocking experiments confirmed HLA-DR+ Teff cells, but not the fraction depleted of HLA-DR+ effectors, to be resistant to Treg suppression mediated via CCR5 and PD-L1 associated pathways. In the presence of HLA-DR+ Teff cells, activation of NFκB downstream of CCR5 and PD-L1 was perturbed. In addition, HLA-DR+ Teff cells expressed significantly higher levels of Th1/Th17 cytokines that may regulate Treg function through a reciprocal counter-balancing relationship. Taken together, our study provides novel insight on how activated HLA-DR+CD4+ T cells may contribute to disease associated inflammation by compromising Treg-mediated suppression in PTB.

Deep sequencing of near full-length HIV-1 genomes from plasma identifies circulating subtype C and infrequent occurrence of AC recombinant form in Southern India.

India has the third largest number of HIV-1-infected individuals accounting for approximately 2.1 million people, with a predominance of circulating subtype C strains and a low prevalence of subtype A and A1C and BC recombinant forms, identified over the past two decades. Recovery of near full-length HIV-1 genomes from a plasma source coupled with advances in next generation sequencing (NGS) technologies and development of universal methods for amplifying whole genomes of HIV-1 circulating in a target geography or population provides the opportunity for a detailed analysis of HIV-1 strain identification, evolution and dynamics. Here we describe the development and implementation of approaches for HIV-1 NGS analysis in a southern Indian cohort. Plasma samples (n = 20) were obtained from HIV-1-confirmed individuals living in and around the city of Bengaluru. Near full-length genome recovery was obtained for 9 Indian HIV-1 patients, with recovery of full-length gag and env genes for 10 and 2 additional subjects, respectively. Phylogenetic analyses indicate the majority of sequences to be represented by subtype C viruses branching within a monophyletic clade, comprising viruses from India, Nepal, Myanmar and China and closely related to a southern African cluster, with a low prevalence of the A1C recombinant form also present. Development of algorithms for bespoke recovery and analysis at a local level will further aid clinical management of HIV-1 infected Indian subjects and delineate the progress of the HIV-1 pandemic in this and other geographical regions.

Circulating Mycobacterium tuberculosis DosR latency antigen-specific, polyfunctional, regulatory IL10+ Th17 CD4 T-cells differentiate latent from active tuberculosis.

The functional heterogeneity of T cell responses to diverse antigens expressed at different stages of Mycobacterium tuberculosis (Mtb) infection, in particular early secreted versus dormancy related latency antigens expressed later, that distinguish subjects with latent (LTBI), pulmonary (PTB) or extrapulmonary (EPTB) tuberculosis remains unclear. Here we show blood central memory CD4 T-cell responses specific to Mtb dormancy related (DosR) latency, but not classical immunodominant secretory antigens, to clearly differentiate LTBI from EPTB and PTB. The polyfunctionality score integrating up to 31 DosR-specific CD4 T-cell functional profiles was significantly higher in LTBI than EPTB or PTB subjects. Further analysis of 256 DosR-specific T-cell functional profiles identified regulatory IL10 + Th17 cells (IL10+IL17A+IL17F+IL22+) to be significantly enriched in LTBI; in contrast to pro-inflammatory Th17 cells (IFNγ+IL17A+/IL10-) in the blood and lung of EPTB and PTB subjects respectively. A blood polyfunctional, Mtb DosR latency antigen specific, regulatory, central memory response is therefore a novel functional component of T-cell immunity in latent TB and potential correlate of protection.

Circulating nucleic acids: An analysis of their occurrence in malignancies.

Through a regulated or fortuitous phenomenon, small portions of cell nucleic acids are thrown into circulation. Since the discovery of these circulating nucleic acids (CNAs) in 1948, numerous studies have been published to elucidate their clinical implications in multifarious diseases. Scientists have now discovered disease-specific genetic aberrations, such as mutations, microsatellite alterations, epigenetic modulations (including aberrant methylation), as well as viral DNA/RNA from nucleic acids in plasma and serum. CNAs have become increasingly popular due to their potential for use as a liquid biopsy, which is a tool for non-invasive diagnosis and monitoring of diseases, such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. While the diagnostic potential of CNAs has been investigated extensively, there is a paucity of understanding of their pathophysiological functions. Are these CNAs part of the cell's regular framework of functioning? Or do they act as molecular players in disease initiation and progression? The aim of this review is to investigate the origins and functions of the circulating cell-free nucleic acids in the plasma and serum of patients with various malignancies, and propose areas of study, which may elucidate the novel underlying mechanisms that are functioning during cancer initiation/progression.

Unbiased Identification of Blood-based Biomarkers for Pulmonary Tuberculosis by Modeling and Mining Molecular Interaction Networks.

Efficient diagnosis of tuberculosis (TB) is met with multiple challenges, calling for a shift of focus from pathogen-centric diagnostics towards identification of host-based multi-marker signatures. Transcriptomics offer a list of differentially expressed genes, but cannot by itself identify the most influential contributors to the disease phenotype. Here, we describe a computational pipeline that adopts an unbiased approach to identify a biomarker signature. Data from RNA sequencing from whole blood samples of TB patients were integrated with a curated genome-wide molecular interaction network, from which we obtain a comprehensive perspective of variations that occur in the host due to TB. We then implement a sensitive network mining method to shortlist gene candidates that are most central to the disease alterations. We then apply a series of filters that include applicability to multiple publicly available datasets as well as additional validation on independent patient samples, and identify a signature comprising 10 genes - FCGR1A, HK3, RAB13, RBBP8, IFI44L, TIMM10, BCL6, SMARCD3, CYP4F3 and SLPI, that can discriminate between TB and healthy controls as well as distinguish TB from latent tuberculosis and HIV in most cases. The signature has the potential to serve as a diagnostic marker of TB.