Abstract
Tumor abnormal protein (TAP) test also called abnormal glycoprotein chain test assesses the level of abnormal glycosylation in the body by measuring the agglutination of 10 different agglutinins, including wheat germ agglutinin, cuttle bean agglutinin, and so on. The lectins are proteins containing one or more binding sites with a strong affinity for particular carbohydrate chains that can specifically identify and bind to abnormal glycan molecules on malignant cells. It has been widely used clinically in recent years for the early diagnosis of tumourigenesis. Numerous studies have been conducted to investigate the mechanisms by which lectins bind to a set of glycans. As the incidence of head and neck cancer is high, with squamous cell carcinoma being the most common type. The lack of highly specific and sensitive tests makes early screening difficult, and treatment is often delayed, resulting in organ loss or even death, and often has a negative psychological impact. This narrative review will analyze the principle and current status of clinical application of TAP detection to prove TAP test will offer more sensitive methods for the precancerous risk assessment of head and neck squamous cell carcinoma, as well as for tracking metastases and recurrence.
Introduction
Approximately 600,000 new cases of malignant tumors of the head and neck are reported worldwide each year, ranking as the seventh most common malignancy. 1 There are various head and neck tumor types, among which squamous cell carcinoma accounting for 90% of cases, with nasopharyngeal carcinoma being the most common and laryngeal cancer being the second, and lymphomas comprising 3%, with the tonsils the organ most often affected. Because early symptoms are easily overlooked, tumors located in the larynx and pharynx are difficult to detect early, and >60% of patients have already developed mid to late-stage disease at the time of first consultation. 2 Although comprehensive treatment, mainly surgery, radiotherapy, and targeted therapy, has greatly extended patient survival, the overall survival rate has not increased significantly in recent decades, with a 5-year survival rate of only 40% to 50%. 3 In addition, surgery for head and neck cancer may result in various post-operative complications affecting facial aesthetics and even resulting in organ loss, while long courses of radiotherapy can make patients resistant to treatment and often have a negative psychological impact. Therefore, more sensitive and accurate risk assessment of precancerous lesions and tracking of tumor recurrence/metastasis are among the most urgent issues that need to be addressed. Proteins on the cell membranes become abnormally glycosylated when malignant changes occur, producing aberrant glycoproteins and changing the structure of their surface glycoconjugates. 4 When their production surpasses a specific threshold, aberrant glycoconjugates can be detected in the peripheral blood. Lectins can specifically recognize and bind to glycans on various glycoproteins through their carbohydrate recognition domains (CRDs). For example, the Ricinus communis agglutinin binds exclusively to carbohydrate chains ending in Galβ1,4GlcNAc, as well as to α,β-D-galactose. 5 Lectin assays, such as TAP (tumor abnormal protein) test, have been reported to have early risk assessment and prognostic value. TAP test can be used for the screening of digestive tract precancerous lesions. 6 For gastrointestinal tumors, 7 as well as breast and lung cancers.8,9 However, the diagnostic utility of these tests for head and neck tumors has received less attention. In this narrative review, we searched from PubMed and introduce the principles underlying lectin detection, describe progress in TAP test in patients with head and neck squamous cell carcinoma, and summarize available detection techniques.
Principle of the TAP test
Lectin structures and the process of glycosylation in normal cells
Lectins are a class of proteins that bind specifically to carbohydrate chains. As lectins contain one or more CRDs and have a high affinity for specific carbohydrate chains, they can cross-link to large numbers of cells, resulting in agglutination of tumor cells. Lectins are excellent probes for research into cell structure because they are selective in their binding to carbohydrate chains and can be used to analyze a range of glycan molecules on the surfaces of cells. 10 Various plant lectins are frequently used to identify carbohydrate chains, 11 including wheat germ agglutinin (WGA), peanut agglutinin (PNA), and lens culinarian agglutinin (LCA).
Proteins are formed in organisms through processes including replication, transcription, and translation, but require further modification after translation to perform their biological functions. Protein post-translational modification (PTM) is a covalent process that occurs in proteins during or after translation through the addition or removal of one or more functional groups, and has a profound effect on protein function. 12 Protein modification processes include phosphorylation, acetylation, ubiquitination, methylation, and glycosylation. 13 Glycosylation is one of the most common PTMs, in which polysaccharides are transferred to specific amino acid residues in proteins by glycosyltransferases. 14 Proteins are modified by glycosylation to produce glycoproteins. Glycoproteins have branched oligosaccharide chains, mostly consisting of 2 to 10 monosaccharides. 15 Glycan molecules can bind to target proteins and perform functions including maintaining protein structure stability and biological activity, regulating intracellular transport, cell activation and apoptosis, influencing cell adhesion, participating in intercellular recognition, antigen and antibody recognition, and regulating signal transduction, among other functions.16,17
Mechanism of aberrant glycosylation in tumorigenesis
Glycoproteins control the interaction of connective tissue with stromal cells and immune cells, and they alter cell differentiation. 18 Abnormal protein glycosylation leads to underexpression, overexpression, and structural abnormalities in glycoproteins, which causes cellular dysfunction and affects the stability of intercellular adhesion proteins. Membrane proteins that have abnormal glycosylation trigger oncogenic signaling cascade reactions, which lead to harmful cell signaling and promote tumor cell proliferation, invasion, and metastasis. The process of glycoconjugates is disrupted in head and neck cancer cells, including inactivation of enzymes involved in the glycosylation process (glycosyltransferases, glycosidases) or activation of enzymes that are already dormant,19,20 resulting in the invasion of cancerous keratinocyte cells into connective tissue.
Application prospects of TAP test by lectin detection in clinical oncology
As we summarized earlier, due to the high affinity between lectins and tumor-specific proteins, they are often used in clinical settings to assist in diagnosing tumors. Tumor development typically involves a progression from precancerous lesions to malignant, recurrence, and even distant metastasis. In light of this, we can investigate the potential of using lectins to predict the occurrence, progression, and prognosis of tumors. TAP test is based on this principle. At present, the TAP detection used in clinical practice includes the detection of 10 lectins. Table 1 elaborates on these ten lectins and the abnormal glycoprotein chain they bind to (Table 1). For example, we can use lectins to predict the probability of malignancy in precancerous lesions, such as leukoplakia and erythroplakia. Early detection in most of the oral cancers is easy as compared to other sites like the larynx, and pharynx due to the early visible changes that might occur in an existing precancerous lesion if performed with an experienced oral diagnostician and with gold standard diagnosis with histopathology. It has been confirmed that the expression of Galectin-3 and E-selectin in leukoplakia is higher than that in normal mucosa, and shows an elevating trend with the malignant transformation of leukoplakia.21,22 Another study has confirmed that glycoconjugates specific to Jack fruit lectin (JFL) and PNA are expressed at different clinical and pathological stages of oral mucosal tumor progression. Mild staining can be detected before the tissue undergoes malignant transformation, while strong staining can occur during malignant transformation, indicating that TAP test can predict the risk of malignancy. 23 At the same time, TAP test can also be applied to predict cervical lymph node metastasis ability and tumor recurrence. In addition to serving as a tumor marker, lectins can also affect the infiltration and metastasis ability of tumors. 4 Kim et al. 24 found that high galectin-1 expression in peritumoral stroma was significantly correlated with depth of invasion in cervical lesions and lymph node metastasis of cervical cancer by transferring gelatin siRNA to cervical cancer cell lines. Versican, a member of the aggrecan gene family, is a large chondroitin sulfate proteoglycan carrying several active domains that enable versatile interactions in a variety of biological and pathological processes. It has multiple active domains that allow it to interact with several physiologic and pathological processes. Versican's position as a clinically highly intriguing tumor progression-related molecule is highlighted by Banerjee's suggestion that its development in premalignant oral lesions may be a possible indication of invasiveness and impending malignant tumor behavior. 25 Pukilla et al. 26 discovered versican expression did not correlate with clinicopathological factors or tumor cell proliferation. In univariate analyses, a higher risk for disease recurrence was associated with a higher stromal versican expression score.
The 10 lectins in TAP test and carbohydrate binding specificity of lectins.
However, some studies showed the protective effect of certain lectins. Immunohistochemical analysis of 243 patient samples showed that the positive staining rate for galectin-7 and S100A9 gradually decreased from normal cervical tissue to intraepithelial neoplasia and cervical squamous carcinoma. 27 Depending on the histological type of the tumor, galectin-7 is a dual regulator of tumor development with possible impacts on cancer cell invasion. 28 Galectin-7 performs its protective effect by upregulating MMP-9 (matrix metalloproteinase-9) expression and cell invasion in lymphoma and cervical adenocarcinoma cells. 29 Tetranectin, a member of the C-type lectin superfamily, was initially implicated as a cancer biomarker because decreased plasma levels of tetranectin correlated with cancer progression. 30 It was detected down-expression in both serum and saliva in metastatic oral squamous cell carcinoma (OSCC). 31 It was speculated that tetranectin may accelerate the activation of plasmin by binding to plasminogen, which is important for the breakdown of extracellular proteins and the development of cancer. 32 It is interesting to note that tetranectin can be detected with a high degree of precision in the saliva of patients with OSCC. 31 Tetranectin levels in patients with metastatic tumors are lower than those in patients with primary tumors, possibly because of significant tetranectin consumption in the tumor microenvironment, where proteolytic activity is required for tumor metastasis. Therefore, challenges that we will investigate in the future include developing more efficient and affordable sample-collecting methods and treating cancers by upregulating the expression of advantageous lectins to lessen recurrence and metastasis.
Current status of TAP in head and neck squamous cell carcinoma
Several lectins have been found to recognize carbohydrate chains in head and neck malignancy cells
The list of lectins that have been found to identify aberrant carbohydrate chains in head and neck carcinoma cells is shown in Table 2. Wong et al. 33 used concanavalin A to bind the N- and O-glycosylated proteins of OSCC cells glycoproteins. They discovered that apolipoprotein A-1 and N-glycosylated α1-antitrypsin, both of which have N-glycosidic linkages, were independent risk factors for the development of OSCC. Baeten et al. 34 discovered that optical imaging analysis of OSCC tissue by fluorescently labeled WGA identified both sialic acid residues and terminal N-acetylglucosamine. Feinmesser et al. 35 demonstrated the presence of PNA binding sites on the cell membrane of laryngeal cancer cells. Chan et al. 36 discovered that lentil from green speckled lentil seeds, not only specifically binds to nasopharyngeal cancer cells (NPCCs), but also resists tumor cell proliferation and promotes apoptosis in NPCCs. Chew et al. 37 demonstrated that the presence of glycoconjugates in the outer parts of NPCCs with terminal sialic acid, Fucose residues, and Gal-D-Gal-Nac residues were altered. Dictyostelium agglutinin and PNA have a strong affinity for NPCCs and specifically bind to abnormally expressed carbohydrate chains. The cancerous characteristics of thyroid cancer cells changed significantly when Kaptan et al. 38 used Maackia amurensis lectin II, Sambucus nigra agglutinin, and Aleuria aurantia lectin to specifically bind to glycans rich in α-2,6, α-2,3, sialic acid and α-1,6 fucose residue on the surface of thyroid cells.
Several identifiable lectins for glycochains have been reported in head and neck malignancies.
The advantages and disadvantages of using TAP test to diagnose head and neck squamous cell carcinoma
The commonly used clinical methods for tumor diagnosis and screening are mainly pathological diagnosis, imaging examination, endoscopy, and tumor marker tests. Pathological diagnosis is the gold standard for tumor diagnosis, but this method is invasive and may cause pain, bleeding, and even tumor spread in patients during biopsy. Carlin et al. 39 reported a case of tumor implantation of hepatocellular carcinoma cells (HCC) in the needle tract, 16 months after a percutaneous fine needle aspiration cytology. Voravud et al. 40 presented two cases in which the spread of cancer cells caused by needle aspiration biopsies turned local, resectable lung cancer into unresectable lung cancer. It is also challenging to obtain materials in some parts, and patients are frequently reluctant to cooperate with throat biopsies, necessitating multiple examinations under general anesthesia. Imaging and endoscopy are the main means of tumor screening, but they are less accurate for early-stage tumors. One of the most common clinical methods for tumor detection is the tumor marker test. Its principle is to detect abnormal glycoproteins in different tumor cells in serum to determine the presence of tumors, a molecule produced by tumor cells themselves or by the body's reaction to tumor cells during the process of tumor occurrence and proliferation, including proteins, mutated genes, and enzymes. Alpha-fetoprotein (AFP) has been used to identify liver cancer and track its prognosis, 41 carbohydrate antigen 125 (CA125) can identify ovarian cancer, 42 and carcinoembryonic antigen (CEA) can identify colon cancer. 43 For precancerous screening, risk assessment, cancer progression, and as a predictor of prognosis, the sensitivity and specificity of this assay are insufficient since it requires the acquisition of high concentrations of glycoproteins. On the contrary, lectin has one or more CRDs on its surface that can specifically recognize and bind glycoconjugates and have a strong binding ability to them. Wong et al. 44 discovered that the degree of TAP protein positive expression is adversely connected with the curative effect of recent radiation on patients. The factor can be used to forecast the short-term curative effect of the molecular markers when TAP high protein is present in individuals with recent bad radiation effects. The presence of abnormal glycoproteins can be determined by detecting TAP and glycoconjugates in serum, 45 even in saliva 46 and urine. 47 Such as galectin-3, which can be detected in serum and urine. 48 Saliva or urine collection is noninvasive, the technique is simple, and the sample collection and preservation are relatively easy. 49 TAP test would be more practical and convenient with the development of tests for lectins in saliva and urine. Tumor cells can be detected in serum when they reach 1 × 108 in the organism, which provides the advantages of convenient, rapid, and noninvasive detection. 50 Therefore, the sensitivity of the TAP test is higher than that of the traditional tumor marker assay, and it is suitable for application in precancerous risk assessment, early cancer screening, recurrences, and metastatic lesions.
However, the range of single-lectin detection is constrained because a tumor may selectively bind to more than one lectin at once. The clinical technique of combining numerous lectins to identify TAP test has been widely utilized, although this approach also raises the false-positive test rate. Shi et al. 51 administered the TAP test to 2363 people who had undergone physical examinations and discovered that the normal population had a positive test rate of 13.97%. In normal and high-risk populations, Jin et al. 52 observed that its positive rate was 2.3% and 23.80%, respectively, but in 160 high-risk individuals with negative TAP expression, prolonged follow-up verified that these individuals did not acquire cancers. This suggested that for the high-risk population, the negative predictive value of TAP is more significant than the positive predictive value. In addition, another disadvantage of the TAP test is that it cannot specifically identify a certain type of tumor cell and therefore cannot accurately locate the primary site of the tumor, so clinical work still needs to combine imaging tests and the patient's specific clinical characters to decide whether to continue the examination or follow-up.
Conclusions
Lectins specifically recognize and bind to carbohydrate chains in abnormal glycoproteins. The cohesive parts that they mix to generate can be observed in patients’ saliva, urine, and peripheral blood tests. The advantages of this test are that it is safe, noninvasive, and highly sensitive. TAP test have more meaningful negative predictive values than positive predictive values in high-risk groups and are particularly suitable for head and neck tumors without ideal tumor markers. These tests provide a highly sensitive and accurate test for precancerous screening, and risk assessment, in second primaries, recurrences, and metastatic lesions of head and neck cancer, gaining time for early treatment of tumor patients.
Although lectins are specific to some tumor cells, the current clinical TAP test methods are still limited to the combined detection of abnormal sugar chains by multiple lectins, which cannot specifically identify a single type of tumor cell, nor can they precisely target the site of tumor production. Clinical studies still need to be combined with imaging tests and patient specificity to decide whether to proceed with testing or follow-up. More research is required on the clinical importance of TAP test in head and neck tumors, the connection between the size of the cohesive fraction created by their binding, pathological and pathological stage, and the possibility of using changes in the cohesive fraction before and after treatment to detect tumor recurrence.
Footnotes
Author contributions
LT wrote the manuscript and collated the articles. LS consulted the literature, participated in the drafting of the manuscript. MW provided oversight for the project and was responsible for conducting the manuscript evaluation. All authors read and approved the final manuscript.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Liaoning Provincial Department of Education Project (JYTMS20230575).
