Uveal melanoma is the most frequent intraocular tumor, with a high mortality rate due to metastases, especially liver metastases. Glycosylation represents an enzymatic process of addition of molecules of saccharides to other saccharides, lipids or proteins. Aberrant glycosylation is considered a key feature in malignant transformation and cancer progression. In uveal melanoma, the most common glycosylation changes are considered sialylation, fucosylation, and N- and I-glycan branching. The goal of this study is to identify sialic acid as a tumoral marker and the correlation with survival rate in uveal malign melanoma. The determination of sialic acid is used in other cancers to monitor the evolution of the disease and the survival rate. Sialic acid specificity is relatively low, because high levels of sialic acid-rich glycoproteins are encountered in inflammatory diseases.
Melanomul uveal este cea mai frecventă tumoră intraoculară, cu o rată mare de mortalitate din cauza metastazelor, în special a celor hepatice. Glicozilarea reprezintă un proces enzimatic de adăugare a moleculelor de zaharide la alte zaharide, lipide sau proteine. Glicozilarea aberantă este considerată o caracteristică-cheie în transformarea malignă şi progresia cancerului. În melanomul uveal, cele mai frecvente modificări ale glicozilării sunt considerate a fi sialilarea, fucozilarea şi ramificarea N- şi I-glicanilor. Scopul acestui studiu este de a identifica acidul sialic ca marker tumoral, alături de corelarea cu rata de supravieţuire în melanomul malign uveal. Determinarea acidului sialic este utilizată în alte tipuri de cancer pentru a monitoriza evoluţia bolii şi rata de supravieţuire. Specificitatea acidului sialic este relativ scăzută, deoarece nivelurile ridicate de glicoproteine bogate în acid sialic sunt întâlnite în bolile inflamatorii.
Sialic acid is a term used to describe derivatives of neuraminic acid, where the amino group of neuraminic acid is substituted by either an acetyl or glycolyl group. The unique structural features of the molecule, which includes a negative charge owing to a carboxyl group, enables it to play a role in cellular functions, such as transport of positively charged compounds, cell-to-cell repulsion, influencing conformation of glycoproteins on cell membranes, and even masking antigenic determinants on receptor molecules.
Sialic acid has a fundamental role in many diseases, including viral infections, cardiovascular diseases, neurological disorders, diabetic nephropathy, and malignancies.
The focus on sialic acid as a tumor marker should be examined from the perspective of aberrant glycosylation in cancer cell membranes owing to activation of new glycosyl transferases that are characteristic of tumor cells, and the role played by sialic acid in tumor cell metastasis, including increased capacity to adhere to vascular endothelium and decreased capacity of cancer cells to be destroyed by host defence mechanisms. The high sensitivity of sialic acid as a tumor marker has been reported in a variety of cancerous conditions(1).
The human red blood cell is studded with nearly 20 million molecules of sialic acid on the outer cell membrane which contributes to its electronegative charge (zeta potential), and by cell-to-cell repulsion prevents red blood cells from aggregating. Owing to its negative charge, sialic acid can bind positively charged molecules and, thus, plays a role in the transport of such molecules. Since they are an essential component of cell surface receptors for a wide range of endogenous (such as peptide hormones) and exogenous (bacteria and viruses) substances, their presence is both a blessing, as well as a scourge. Thus, they have a role in the cellular actions of hormones such as insulin and, also, they can modulate amino acid transport in some cells. On the other hand, infection by bacteria or virus is solely dependent on the presence of sialic acid as a component of specific receptors for the microorganisms on the cell membrane(2).
As antigenic determinants of glycoproteins or glycolipids, sialic acid molecules contribute to the specificity of blood group substances. The negative charges on sialic acid have an influence on the conformation of glycoproteins in terms of their proper alignment in cell membranes, the expression of enzymatic activity of glycoprotein enzymes, and even resistance to proteolytic enzyme degradation. Indeed, the clustering of cell membrane glycoproteins owing partly to the repulsion of their oligosaccharide sialic acid residues, is important for cell rigidity, since the loss of sialic acid molecules can increase the deformability of cells(3).
An intriguing role for sialic acid is its ability to serve as biological masks by preventing ligands from recognizing receptors. Thus, a glycoprotein layer rich in sialic acid acts as an immune barrier between the mother and the fetus. Indeed, this masking effect is lost by the removal of terminal sialic acid residues from oligosaccharide chains, since it leads to the exposure of a penultimate galactose residue which is then recognized and bound by naturally occurring antibodies, thus facilitating the removal of glycoprotein or the cell by the reticuloendothelial system.
The relevance of sialic acid to the tumor cell is apparent from the increased sialylation and sialyltransferase activity observed in many cancer cells(1).
The aberrant glycosylation found in cancer cell membranes is presumably due to the activation of new glycosyltransferases that are characteristic of tumor cells and are absent or present only in small quantities in normal cells(4).
Thus, for instance, a relatively specific sialyltransferase is found to be present by as much as 2.5 to 11 times in greater amounts in transformed cells when compared to control cells(5).
Sialic acid bound to membrane glycoproteins, and glycolipids apparently enters the circulation by either shedding or by cell lysis. Approximately 98 to 99.5 percent of total sialic acid found in serum or plasma is bound to glycoproteins. Only a very small fraction of sialic acid is bound to lipids which is mainly in the form of gangliosides(1). Normal levels of total sialic acid in serum are approximately in the range of 51 to 84 mg/dL. In contrast, the contribution of the pure lipid fraction to the total sialic acid level is barely in the range of 0.4 to 0.9 mg/dL(6).
Glycosylation is the enzymatic process that produces glycosidic linkages of saccharides to other saccharides, lipids or proteins(7).
Glycosylation is a frequent and well known post-translational protein modification, and probably much more frequent than phosphorylation. The glycome, or complete pattern of glycan modifications in a cell or tissue, is assembled by the synchronized action of numerous glycan modifying enzymes. These enzymes include glycosyltransferases and glycosidases that glycosylate various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. How much a given protein is glycosylated depends on the presence and frequency of glycosylation sites in the protein sequence, as well as the expression and activities of specific glycosylation enzymes within the cell or tissue(8).
The most widely occurring glycosylation events in melanoma are sialylation, fucosylation, and N- and I-glycan branching(9).
Sialylation
Sialylation is an enzymatic process that consists in the covalent addition of sialic acid to the terminal position of glycan chains on glycoproteins and glycolipids. The synthesis of sialylated glycans is catalyzed by the sialyltransferase family, which consists of 20 subtypes in humans. All of them can produce both N-linked and O-linked glycans, which are involved in several cellular processes. Golgi-located or membrane-bound sialyltransferases use cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) as a donor to catalyze the formation of a glycosidic linkage between C2 of the acid sialic from the donor and C3, C6 or C8 hydroxyl of a glycan acceptor. Accordingly, sialyltransferase subtypes are named ST3, ST6 or ST8(9).
The link between melanoma progression and aberrant sialylation is well established. Increased á2,3-sialylation is associated with a more aggressive phenotype in melanoma cells. In particular, the presence of aberrant sialoglycoconjugates on á5â1 integrin has been shown to correlate with a more aggressive phenotype in melanoma cell lines(10).
A recent study from our group reported that the expression of the á 2,3-sialyltransferase ST3GAL1 correlates with melanoma progression and highlighted the critical role of ST3GAL1 in driving melanoma metastasis(11).
Silencing of this sialyltransferase suppressed melanoma migration and invasion and reduced the ability of aggressive melanoma cells to enter the bloodstream, colonize distal organs and survive in the metastatic environment. Further, this study shed light on the regulation of ST3GAL1 in melanoma cells. It was found that the oncogenic transcription factors GLI1 (one of the final mediators of the Hedgehog pathway) and SOX2 (a known pluripotency transcription factor) co-regulate ST3GAL1 transcription. Moreover, the tyrosine kinase receptor AXL was identified as a ST3GAL1 substrate, which is fundamental for the pro-invasive effects of ST3GAL1 in melanoma cells(9).
Recent evidence highlighted the role of sialic acids on the surface of cancer cells to protect them from destruction by the immune system(12) in several types of cancer, including melanoma. Perdicchio and colleagues have first shown that hypersialylation of B16 melanoma cells positively affects tumor growth by facilitating the escape from the immune system at multiple levels(13).
Reduction of sialic acids on B16 cells by silencing the CMP-sialic acid transporter Slc35a1 slowed down tumor growth in vivo and promoted an anti-tumor immune response, boosting the tumor infiltration of effector T cells and decreasing the frequency of T regulatory cells(9).
Perdicchio and colleagues also provided evidence that the altered Treg/Teff balance in this model depends on the increased influx and activity of natural killer (NK) cells, as their depletion abolished the induction of anti-tumor immunity. Thus, reducing sialylation could provide a therapeutic strategy to make melanoma cells more susceptible to immune attack(13).
Dendritic cells (DCs) represent a class of immune cells which offer good prospects for anti-cancer immunotherapy(14).
Human DCs display a high content of sialic acids, which inhibits their maturation and co-stimulation ability. Silva and colleagues have shown that desialylation of DCs improves their ability to elicit T cell-mediated anti-tumor activity(15).
Desialylation of human DCs induced their maturation, increased expression of major histocompatibility complex I and II (MHC-I and -II) and co-stimulatory molecules, and stimulated IL-12 secretion. Furthermore, desialylated DCs displayed increased peptide presentation through MHC-I, leading to the activation of CD8+ cytotoxic T cells. Accordingly, desialylated DCs in co-culture with OVA-expressing B16 melanoma cells improved T cell-mediated cytotoxicity against tumor cells. Therefore, sialidase treatment of DCs might represent a novel tool to improve the efficacy of DC-based anti-cancer vaccines(15).
Haas and colleagues provided evidence that melanoma-intrinsic hypersialylation suppresses effector functions of Siglec-9+ CD8+ T cells in the tumor microenvironment (TME)(16).
RNA-sequencing data from the TCGA melanoma database revealed that the sialyltransferases ST3GAL5, ST6GALNAC2 and ST3GAL6 are consistently expressed in the investigated melanoma samples. As a result, enzymatic digestion of Siglec-9 ligands on target cells by neuraminidase only enhanced the cytotoxicity of the Siglec-9-expressing cells. Thus, key effector functions of CD8+ T cells are abrogated by Siglec-9 engagement, which is related to the phosphorylation of the inhibitory protein phosphates SH-P1 in vitro. The reported findings show that selected sialyltransferases are implicated in the biosynthesis of Siglec-9 ligands, which exert an immunosuppressive role in melanoma progression(16).
Fucosylation
Fucosylation is a type of glycosylation that catalyzes the attachment of fucose sugar units to a molecule. Fucosylation is performed by fucosyltransferase (FUT) enzymes. Fucosylation of glycoproteins is one of the most important features that mediate several specific biological functions in normal and cancer cells(9).
FUT is a group of enzymes that catalyze the incorporation of fucose from activated nucleotide donor GDP-fucose to the reducing end of complex glycans in a linkage-specific manner. These enzymes are widely expressed in many different tissues(17).
Thirteen fucosyltransferase genes have been identified in the human genome. These can be further classified into three subfamilies: á-1,2 FUT, á-1,3/4 FUT, and á-1,6 FUT(18,19).
Fucosylation can be divided into terminal and core fucosylation. Among FUT enzymes, FUT8 is an á-1,6 fucosyltransferase and the only FUT responsible for core fucosylation on N-glycoproteins, as most of the other fucosyltransferases are functionally redundant(18,20-22).
In cancer, fucosyltransferases play an important role in the biosynthesis of tumor-associated antigens, including Lewis (Le)a and (Le)b, sialyl Lewis A (sLe)a and sialyl Lewis X (sLe)x, as well as the H blood group antigen(17).
One of the first pieces of evidence that fucosyltransferases might be implicated in melanoma progression came from the finding that the expression levels of FUT1 and FUT4 mRNA are significantly higher in metastatic melanoma cell lines (A375, WM9, WM239) compared to primary melanoma cells (WM35)(23).
LTA (Lotus tetragonolobus agglutinin), the lectin that specifically recognizes fucose residue of glycans and L-fucose, reduced adhesion to fibronectin and collagen IV of all primary and metastatic cell lines and slightly decreased the proliferation of metastatic but not primary melanoma cell lines(23).
A more recent report confirmed the importance of FUT4 in migration and invasion of human melanoma cells that occur through the activation of the PI3K/AKT signaling pathway(24).
Other studies pointed toward a role of FUT4 in regulating melanoma cell growth. It has been shown that NF-êB/p65-dependent transcriptional regulation of FUT4 modulates human melanoma cell proliferation(25).
Administration of the ginsenoside Rg3, a herbal medicine with anti-tumor activity, suppresses the growth of human melanoma xenografts by decreasing the expression levels of FUT4 and p65 in vivo(25).
Another report showed that Rg3 inhibits melanoma cell growth through inhibition of EGFR phosphorylation and FUT4/LeY downregulation in vitro and in vivo(26).
These studies suggest that suppression of FUT4 expression/activation by Rg3 may be a potential therapeutic strategy for melanoma treatment(9).
Agrawal and colleagues performed a systematic analysis of the melanoma glycome of clinical samples and found upregulation of core fucosylation (FUT8) and downregulation of á-1,2 fucosylation (FUT1, FUT2) as features of metastatic melanoma(27).
The pro-metastatic role of FUT8 was confirmed in vitro and in vivo(9).
FUT8 silencing decreased cell invasion and in vivo melanoma metastasis, suppressing the ability of melanoma cells to colonize distant organs. In addition, suppression of FUT8 impaired the growth of established metastasis in vivo, using a model based on intracardiac instillation of tumor cells(9).
The increase in FUT8 expression in melanoma cells was found to be transcriptionally regulated by TGFâ-induced factor homeobox 2 (TGIF2)(27).
Proteomic analysis of core-fucosylated proteins identified several regulators of invasion and metastasis, including the L1CAM(9).
This cell adhesion molecule is cleaved by plasmin, and the proteolytic cleavage inhibits its ability to mediate spreading and metastasis(28).
FUT8-mediated core fucosylation prevents L1CAM cleavage by plasmin, facilitating melanoma cell invasion. The ability of uncleaved L1CAM to interact with the vasculature at distal organs may explain how FUT8 contributes to melanoma metastases(27).
The fucose salvage pathway is a two-step process in which mammalian cells transform L-fucose into GDP-L-fucose, a universal fucose donor used by fucosyltransferases to modify glycans(18).
It has been reported that the expression of fucokinase (FUK), a key enzyme in the fucose salvage pathway, is downregulated in metastatic melanoma, limiting GDP-L-fucose substrate availability(29).
The treatment of melanoma cells with L-fucose, or FUK overexpression, resulted in decreased migratory potential as well as increased cell surface fucosylation. The administration of L-fucose not only slowed tumor growth, but also inhibited lung metastases in a melanoma mouse model(29).
A recent report showed that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting á-1,2 fucosylation(30).
The activation of the fucose salvage pathway by ectopically expressed FUK decreased invadopodium numbers and inhibited the proteolytic activity of invadopodia in melanoma cells. The inhibition of invadopodium formation by L-fucose or FUK can be rescued by treatment with á-1,2, but not á-1,3/á-1,4 fucosidase, suggesting an á-1,2 fucose linkage-dependent anti-metastatic effect(9).
As a conclusion, the expression of the á-1,2 fucosyltransferase FUT1 was found downregulated during melanoma progression, and its ectopic expression was sufficient to inhibit invadopodium formation and extracellular matrix degradation in melanoma cells(30).
These reports suggest that the functional consequences of fucosylation in melanoma are likely linkage-dependent; the core-fucosylation (á-1,6 fucosylation) mediated by FUT8 promotes melanoma progression(27), whereas branched fucosylation through the á-1,2 linkage inhibits melanoma invasion and progression(27,29,30).
N- and I-glycan branching
Increased expression of complex â1,6-branched N-linked glycans represents a frequent glycosylation modification occurring in the Golgi membrane during malignant transformation and the acquisition of metastatic potential(31,32).
The enzymatic activity of á-1,6-mannosylglycoprotein 6-â-N-acetylglucosaminyltransferase V (GnT-V), which is encoded by the MGAT5 gene, produces tri-/tetra-antennary N-glycan species that can modulate protein’s half-life, stability, extracellular-binding proteins as well as functional activity(9).
Expression of MGAT5/GnT-V is transcriptionally regulated by several oncogenic inputs, including the transcription factor Ets-1 in several cancer cell lines, including melanoma(33,34), and by the receptor tyrosine kinases Her-2/neu(35) and Src(36).
GnT-V expression can regulate N-glycosylation of Her-2 and Her-2-induced signaling pathways. Knockdown of GnT-V results in inhibited expression of N-linked â-1,6 branching on Her-2 and impaired Her-2-induced signaling pathways, which leads to upregulation of the protocadherin â gene cluster, contributing to reduced Her-2-mediated mammary tumorigenesis(37).
One of the first evidence that GnT-V is involved in melanoma progression came from the finding that hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells display increased GnT-V activity, â1,6 branching in glycoproteins, upregulation of integrin subunits á3, á5, á6, áv, â1 and â3, and increased metastatic potential in vivo and motility in vitro(38,39).
N-glycoproteins bearing GlcNAc â1,6-branched N-glycans were identified in the metastatic A375 human melanoma cell line. These include integrin subunits á2, á3, á5 and â1, as well as N-cadherin and lysosome-associated membrane proteins (LAMP-1 and LAMP-2)(40).
A further study identified proteins bearing â1-6 branched N-glycans in human melanoma cell lines from different progression stages. Mass spectrometry analysis revealed that primary melanoma cells WM35 show the lowest number of proteins possessing â1,6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines(9).
Among identified proteins, the largest group consists of integrin subunits. In addition, L1CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma-associated antigen, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were also found(41).
The increasing amount of GlcNAc â1,6 glycans on á5â1 and á3â1 integrins in metastatic cells plays a role in integrin-dependent migration on fibronectin and likely contributes to their acquisition of metastatic ability(42).
Another study reported that â1,6-branched N-glycans affect FAK signaling in metastatic melanoma cells, enhancing FAK autophosphorylation on Tyr397, and resulting in enhanced migration on vitronectin(43).
Cell surface N-glycans with GlcNAc â1,6 branches have also been shown to contribute to uveal melanoma progression by enhancing cell motility(44).
This could be in part explained by a more abundant presence of GlcNAc â1,6-branched N-glycans and higher expression of MGAT5 in uveal compared to cutaneous melanoma cell lines, contributing to their ability to migrate in fibronectin(45).
Recent findings highlighted the critical new role for blood group I antigens (I-branched glycans) as emerging effectors of cancer progression(46).
Synthesis of I-branched glycans, Galâ1,4GlcNAc moieties linked in â1,6 linkage to galactose residues on fetal i-antigen, is initiated by the master I-branching enzyme GCNT2(47).
This enzyme regulates the conversion of linear poly-LacNAcs normally expressed on fetal and cord blood cells to I-branched glycans found on adult erythrocytes and mucosal epithelia(48,49).
GCNT2/I-branched glycans have shown both positive and negative relationships with cancer progression, depending on the tumor type(46).
A recent study revealed that loss of GCNT2/I-branched glycans in melanomas regulates multiple cell surface glycoprotein signaling pathways and promotes melanoma growth and survival(50).
Sweeney and colleagues demonstrated that, while normal epidermal melanocytes display abundant I-branches, these structures progressively diminish in primary and metastatic melanomas(9).
This finding underlines the inverse correlation between GCNT2 and melanoma progression reported by in silico and immunohistochemical analysis, suggesting that the loss of GCNT2 expression could be used as a biomarker of melanoma(51).
This study clearly showed that the knockdown of GCNT2 significantly enhances melanoma xenograft growth and three-dimensional colony formation and survival, whereas GCNT2 overexpression has the opposite effect(9).
Analyses of two representative N-glycosylated protein families, insulin-like growth factor-1 receptor (IGF1R) and integrins, revealed that GCNT2/I-branched glycan modifications inhibited IGF-1 and ECM-mediated melanoma cell proliferation, survival and associated downstream signaling pathways(50).
It is clear that N- and I-glycan branching are emerging as critical effectors of melanoma and correlate both positively and negatively with melanoma progression, regulating malignant-associated adhesive, migratory, growth, survival and metastatic activities(9).
Materials and method
Aim of the study
This prospective study aims to identify sialic acid as a tumoral marker and the correlation with survival rate in uveal malign melanoma. The determination of sialic acid is used in other cancers to monitor the evolution of the disease and the survival rate. Sialic acid specificity is relatively low, because high levels of sialic acid-rich glycoproteins are encountered in inflammatory diseases. The patients were followed for a period of two years, with serum measurements taken before and after treatment, and at subsequent six-month intervals. The therapeutic decision of radiotherapy or enucleation was performed in each case by the oncological commission based on patients’ parameters, independent of the study.
Study inclusion and exclusion criteria
The inclusion criteria for the study were: age between 18 and 70 years old, confirmed clinical and paraclinical diagnosis of malignant uveal melanoma, Eastern Cooperative Oncology Group (ECOG) scores between 0 and 2. All patients were enrolled after providing the written informed consent for study participation, which could be withdrawn at any point during the study should the patient choose to.
The following patients were excluded from the enrollment into the study population: age outside 18-70 interval, altered clinical status with extensive metastatic disease throughout the body, other complex oncological treatments, patient behest to not participate or withdrawal of consent at any point during the study.
Laboratory determinations methodology: after providing the written consent, venous punction blood samples were taken from the patients. The samples were centrifugated and the resultant serum isolate was used for laboratory determinations.
Statistical softwareMinitab® 20 (Minitab Ltd, Coventry, United Kingdom) and SPSS version 29 (the current, subscription-based version) (International Business Machines Corporation, IBM, Endicott, New York) were used for the statistical analysis of the study data.
Determination of sialic acid in patients with uveal melanoma
The principle of the method
The dosage of sialic acid was adapted according to the method of Kattermann and Kriegel (1981), and is based on oxidation with periodic acid, resulting in the formation of â-formylpyruvic acid, which reacts with two molecules of thiobarbituric acid and results in a pink chromophore, with the maximum absorbance at a wavelength ë= 549 nm.
Reagents
N-acetyl-neuraminc acid, standard 1 mmol/L (Merck, Germany).
Pipette the samples, 0.05 mL into each test tube, then add 0.95 mL of 0.1 N sulfuric acid.
Hydrolyze 0.05 mL of serum with 0.95 mL of 0.1 N sulfuric acid for 1 hour at 80ºC.
Pipette 20 µL of standard solution, over which add 220 µL of 0.1 N sulfuric acid solution.
Pipette 240 µL of the hydrolyzed samples.
Add 100 µL of solution A.
Incubate for 20 minutes at room temperature.
Add 200 µL of solution B.
Stir until the brown color disappears.
Add 1 mL of solution C.
Boil for 15 minutes.
Extract the final dye with 1.6 mL of cyclohexanone.
Centrifuge for 10 minutes at 1200 g.
Extract the supernatant.
The samples are read at 549 nm, on the Specord M40 UV-VIS spectrophotometer.
Calculation of results
A549nmsample/A549nmstandard x 83.3=µmol/L NANA, where NANA=N-acetyl neuraminic acid.
To obtain the final result, the value is multiplied by the dilution of the sample (20) and expressed in mM.
Reference laboratory values provided were set at 1.58-2.22 mmol/L for serum/plasma levels of total sialic acid (TSA), 0.5-3 mmol/L for free form of sialic acid, and 10-50 mmol/L for the lipid-associated sialic acid (LSA) forms. The first determination was taken before starting the treatment and the following at repeated checks every six months.
Results
Sialic acid serum levels were measured at intervals of six months. The first determination was performed pre-treatment (1st Determination), the second determination was performed six months after the treatment (2nd Determination), the third determination 12 months after the treatment, and so forth. In total, 99 1st Determinations were recorded, 44 for the second determination (six-month follow-up), 29 for the third determination (one-year follow-up), 12 for the fourth (one-and-a-half-year follow-up), seven for the fifth determination (two-year follow-up), four for the sixth determination, three for the seventh one, and two for the eighth. The maximal follow-up included was for two patients at three and a half years after the treatment onset. The extended descriptive statistics are presented in Table 1.
We observed a trend for decreasing maximal serum sialic acid (mmol/L) measurement in later follow-up data, with a maximum decrease at the third determination (3.75 and 3.62, versus 3.08 at 3rd Determination) and another decrease from the fifth measurement (5th, 6th and 7th Determinations).
We performed data distribution analysis using the Anderson-Darling Normality test, returning a normal distribution for 3rd-8th Determinations (p>0.05), but not for 1st and 2nd Determinations (Ryan-Joiner). We present a graphical summary of our findings in Figure 1.
A comparison of the mean serum mmol/L sialic acid values recorded in the study was performed against the reference range provided by the laboratory (1.58-2.22 mmol/L), which is presented in Figures 2 and 3.
A comparison between the pre-treatment measurement (1st Determinations) and each subsequent follow-up determination was performed using the nonparametric Mann-Whitney test, yielding a statistically significant difference at the six-month follow-up (1st Determination median 2.14 versus 2nd Determination median 2.16; p=0.03; p<0.05), at the one-year follow-up (1st Determination median 2.14 versus 3rd Determination median 2.24; p=0.047; p<0.05), and the two-year follow-up (1st Determination median 2.14 versus 5th Determination median 2.36; p=0.024; p<0.05). Comparison of the 1st Determination against the 4th, 6th, 7th and 8th Determinations yielded no statistical difference. Overall, serum sialic acid levels were increased post-treatment, with statistical significance at six-month, one-year and two-year follow-up, and afterwards the medium value decreased back into the reference range intervals (Figures 2 and 3).
Discussion
Total lipids and lipids-bound or “lipids-associated” sialic acid levels are increased in a variety of tumors(52-54).
In a recent study on the usefulness of total and “lipid-associated sialic acid” (LASA) in lung cancer, a total of 152 patients with primary lung cancer who had not undergone treatment and 107 patients with benign pulmonary disease were evaluated alongside 207 normal subjects(1).
These data revealed that the mean concentration of total sialic acid (TSA) and LASA were significantly higher in lung cancer patients when compared to benign and normal controls.
At the designated cutoff levels in serum for TSA and LASA, which were respectively 80 mg/dl and 20 mg/dl, the sensitivity or the percentage of true positives for lung cancer was 86.5% for TSA and 77% for LASA(1).
However, the specificity of TSA and LASA were low when compared using as negative control the value obtained on benign pulmonary patients (TSA and LASA had both a specificity of 44%)(1).
LASA levels have been reported to be useful in monitoring patients with malignant melanoma(1).
In one study, when tumor recurrence was correlated with elevated LASA levels, the increased level was found as early as 9.3 months (median value) prior to recurrence(55).
Higher levels of TSA and LASA have been reported in leukemia patients compared to patients with anemia(56).
The TSA levels were significantly higher in acute myeloid leukemia (AML) compared to chronic myeloid leukemia (CML) and acute lymphatic leukemia (ALL) patients(1).
The LASA levels were significantly elevated in AML patients as compared to other leukemic patients. The sensitivity of sialic acid as a marker for leukemia is high, with the sensitivity of LASA approaching to 85%(1).
The TSA levels in patients with oral and maxillofacial malignancy were reported to be significantly higher in patients with cancer stages III and IV, when compared to patients with cancer stages I and II(1).
During the follow-up of response to treatment, while TSA levels declined during remission of disease, they became elevated with recurrence and metastasis(57).
Sialic acid measurements appear to have a high sensitivity for a wide range of tumors(1).
The specificity of sialic acid measurements – especially the nonspecific LASA measurements – is low, since the latter is elevated in benign disorders particularly associated with inflammatory conditions, because in inflammatory conditions the level of acute phase proteins, such as alpha-1 acid glycoprotein, are expected to be elevated, and we can also expect an apparent elevation of LASA(1).
In our study, the data confirm the fact that sialic acid has a low specificity in monitoring the evolution of uveal melanoma. The high levels of sialic acid obtained in the first determinations cannot be correlated only with the evolution of the tumor or the presence of metastasis, because the post-treatment inflammation can also increase the serum levels of sialic acid.
The low values from the last measurements can be correlated with a regression of a neoplastic disease or absence of metastasis, but also with the decrease of inflammation after the oxidative stress induced by the treatment.
Conclusions
The ability of tumors to mimic host-like cell-surface sialylation – in particular, hypersialylation – is a key element of cancer progression and aggressiveness, by enabling evasion of the immunosurveillance system and cell death pathways, while increasing the capacity for metastasis(58).
Hypersialylation of cancer cells also plays a cytoprotective role and contributes to chemotherapy and radiotherapy resistance in several cancers by mechanisms that are still being explored, but believed to be partly due to the added physical barrier of excess sialic acid, both absorbing ionizing radiation and blocking the diffusion of drug molecules into the cell. It has been shown that increased sialylation of cancer cells can confer resistance to chemotherapeutics such as paclitaxel and cisplatin(59-62).
To date, no specific hypersialylation mechanisms that promote radiotherapy resistance have been identified, although correlations have been observed, which implicate the importance of STs in radioresistance, especially in colorectal cancers(63-66).
Further investigation into these sialic acid-related processes is needed to enable the design of effective anti-metastatic agents that target the key sialyltransferase subtypes involved, with further potential to disarm resistance mechanisms(58).
Aberrant glycosylation in uveal melanoma can also be used to develop new therapeutic strategies. Two recent studies identified core fucosylation (FUT8) and the sialyltransferase ST3GAL1 as critical drivers of melanoma metastasis, highlighting the therapeutic potential of targeting FUT8 and ST3GAL1 to treat metastatic melanoma(11,27).
These studies provide a rationale for the future design of small molecule inhibitors against FUT8 and ST3GAL1 to prevent or treat established melanoma metastasis(9).
Due to its low specificity, sialic acid cannot be used as a single marker for the evolution and monitoring of uveal melanoma, because high serum levels of sialic acid may appear in aggressive neoplastic disease or in the presence of metastasis, but also in a high inflammatory state. Determinations of serum sialic acid are needed to be completed with other tumoral markers to obtain more accurate data, therefore further studies are needed to prove its importance.
Autori pentru corespondenţă: Christiana-Diana-Maria Dragosloveanu E-mail: christianacelea@gmail.com
CONFLICT OF INTEREST: none declared.
FINANCIAL SUPPORT: none declared.
This work is permanently accessible online free of charge and published under the CC-BY.
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