Gastric cancer is a heterogeneous disease that requires multidisciplinary treatment [1
]. Currently, new molecular classifications for gastric cancer have been proposed by TCGA (The Cancer Genome Atlas) and ACRG (Asian Cancer Research Group) in which microsatellite instability (MSI) is described as a distinct subgroup [1
]. According to TCGA, the MSI subgroup is linked with hypermutation, gastric CpG island methylator phenotype, MLH1 silencing, and mitotic pathways [1
]. In the last few years, many publications described MSI from a clinical point of view, being found in 5.6% to 33.3% of all gastric cancers and strongly associated with female sex, older age, intestinal histotype, middle/lower gastric position, N0 status, and TNM stage I/II with favorable overall survival [1
]. It is important to underline that MSI is not a homogenous group and it has been shown that MSI is a prognostic factor because of the association with tumor localization and Lauren classification [6
]. Additionally, it has been found that MSI is linked with PD-L1 expression and several specific drugs are currently used for clinical management of gastric cancer targeting the PD1/PD-L1 immune checkpoint pathway [7
]. The molecular diagnosis of MSI gastric cancer is of highest importance, especially before multidisciplinary treatment. It presents different responses to neoadjuvant chemotherapy and requires specific surgical treatment in synchronous metastases, includes the possibility of tailored lymphadenectomy, and stratifies for the application of targeted therapies [3
The transformation of gastric epithelial cells is accompanied by several changes in the protein glycosylation machinery resulting in the aberration of cellular glycosylation, such as the truncation of O
-linked glycans and the expression of sialofucosylated glycan epitopes [9
]. The truncation of O
-linked glycans has been widely described in various gastrointestinal carcinomas and results in the de novo
expression of short O
-glycans such as the Thomsen-Friedenreich (TF or T, Galβ1-3GalNAcα1) antigen, Thomsen-nouvelle (Tn, GalNAcα1) and its sialylated form sialyl Tn (STn, Neu5Acα2–3GalNAcα1) [9
]. In addition, the expression of complex sialofucosylated structures, such as sialyl-Lewis A (SLea
, Neu5Acα2–3Galβ1–3 [Fucα1–4] GlcNAcβ-) and sialyl-Lewis X (SLex
, Neu5Acα2–3Galβ1–4 [Fucα1–3] GlcNAcβ-) has been frequently described in gastric tumors with an important role in cancer progression and metastasis formation [9
The TF antigen, also known as core 1 structure, is an intermediate structure during the maturation of mucin-type O
-glycans within the Golgi apparatus. Under physiologic conditions, the TF antigen is below the limit of detection because it is modified with additional saccharides and made inaccessible due to surrounding larger glycans [10
]. It is only found in the luminal surfaces of the pancreatic duct, kidney distal tubule and kidney collecting duct [11
]. In addition, macrophages of the thymus, spleen and lymph nodes carry the TF antigen, suggesting a potential immunologic role [11
]. Importantly, the TF antigen is neither found in the healthy gastric epithelium nor in gastric pre-malignant conditions [12
]. In gastric cancer the TF antigen is expressed in considerable amounts in around 21% of the tumours, associating with higher immune response [12
As molecules that are secreted into circulation and due to their expression specificity for malignant cells, aberrant glycans and glycoconjugates have a long-lasting history as cancer biomarkers [13
]. We report here the analysis of TF, Tn, STn, SLea
expression in gastric carcinomas in a cohort of 30 patients, 13 with MSI high and 17 with MSI low or negative status, revealing a novel strong association between TF expression and MSI status. Our data suggest the TF antigen as a single specific and sensitive marker for the MSI status.
We analyzed 30 gastric carcinoma cases (13 MSI high and 17 MSS/MSI low) for the expression of TF, STn, SLex
. Additionally, we have previously evaluated this cohort for the expression of Tn [17
]. The expression of each antigen was used for association analyses with MSI status (Table 1
), which revealed a highly significant association between the expression of TF and MSI status (p
< 0.001; Fisher’s exact test). Among the 10 TF positive cases, 9 had MSI, which suggests an unprecedented specificity of the TF antigen for this gastric cancer molecular subtype (Table 1
). Our results indicate that the TF antigen has also very good sensitivity, as 16 of the 20 TF negative cases were MSS or MSI low (Table 1
). This results in sensitivity and specificity values of 69.2% (9/13) and 94.1% (16/17), respectively. Positive and negative predictive values were 90% (9/10) and 80% (16/20), respectively.
The TF epitope was expressed in 10 of the 30 evaluated gastric carcinoma cases. The mucosas adjacent to tumors, including normal glands and highly inflamed regions, as well as intestinal metaplasia and dysplasia, were completely negative, underlining the absence of this glycan marker from non-malignant and pre-malignant conditions (Figure 1
Among the positive cases, the staining was typically membranous and found on average in around 30% of all cancer cells of the tumor. In well-differentiated gastric carcinomas, the staining was typically at the apical membrane and included secretion (Figure 1
d–f). The subcellular localization among poorly differentiated gastric carcinomas shifted typically to cytoplasmic (Figure 1
We did not find any further statistical significant association between the TF expression and other clinicopathological features (Table 2
However, the expression of TF seems to associate with good prognosis for the patients, reflected by the increased median patient survival (88 months vs. 31.5 months) and the high proportion of patients being alive 5 years after diagnosis (70% vs. 30%) (Table 2
). This improved prognosis is in accordance with expectations, as an on average better survival is known to occur for MSI patients.
Here we present for the first time strong evidence that the cellular expression of the TF antigen is associated with the MSI gastric cancer subtype. Due to the high amount of glycoconjugates expressed by cancer cells and secreted into circulation, cancer-specific glycan epitopes represent promising targets for biomarker application in blood tests and liquid biopsies. Indeed, most of the established cancer biomarkers currently applied in the clinical setting detect glycan moieties or glycoconjugates [13
]. In contrast, current MSI analysis is a resource intensive procedure evaluating either the tumor PCR profile of 5 MSI markers and comparing them to the profile of matching normal DNA or evaluating the absence of at least one of four nuclear expressed markers in tumor sections.
Mismatch repair deficiency (dMMR) testing can be performed by immunohistochemistry in paraffin sections using commercially available antibodies evaluating the expression of four MMR proteins (MLH1, PMS2, MSH2 and MSH6) in tumor cell nuclei or by PCR, as mention above. Usually, MMR status is tested in colorectal cancer specimens for the identification of patients at elevated risk for Lynch syndrome as well as for prognostic stratification. However, recent data has emerged showing that dMMR can have predictive value for immune checkpoint inhibitor therapy, regardless of the cancers’ tissue of origin [19
]. In fact, it was approved in the last year by the Food and Drug Administration that pembrolizumab (anti-programmed cell death protein-1 (PD-1)) could be used in any solid tumor with MSI or dMMR that have progressed following prior treatment.
Our data indicate that the TF antigen, as a single marker, is a highly reliable predictor of MSI status in gastric cancer. The application of TF has, therefore, strong potential for the stratification of patients with MSI in a time and cost-efficient manner.
Thus, the TF antigen holds promise to be the long sought-after single biomarker for MSI status in gastric cancer. Moreover, TF has several additional assets which could allow it to be used as serologic biomarker application. Firstly, it is hardly expressed in human tissues, neither under healthy nor under pathologic conditions [11
]. Secondly, the basal levels of exposed TF are very low in the bloodstream due to naturally circulating anti-TF IgM and IgG antibodies and the rapid clearance of terminally galactosylated glycoconjugates by the liver [21
]. Lastly, as a mucin-type O
-glycosylation it may be carried by a wide range of secreted glycoproteins that are overexpressed in gastric cancer such as MUC1 or CD44. Future studies testing the applicability of TF detecting ELISA based serological assays for the diagnosis of MSI subtype have potential utility in the clinical setting. In addition, other strategies such as the detection of elevated levels of autoantibodies against TF, as it has been frequently described in cancer [21
], could have indicative properties for the MSI status.
The TF antigen is a simple glycan antigen that is aberrantly expressed in cancer cells due to alterations in the O
-glycan biosynthesis pathway. However, the underlying molecular mechanism that leads to the expression of the TF-antigen in this molecular subtype and the phenotypic consequences remain elusive. The expression of TF might facilitate the tumor immune response evasion, since the TF epitope is a potent ligand for galectin-1 and -3, which are known to modulate the immune response [22
Another possible explanation is that the TF epitope is a byproduct of increased tandem repetitions within the MSI cancer cell genome. The abrogation of an efficient DNA mismatch repair system leads to the increase and creation of new simple sequence repeats, known as microsatellites. The detection of these sequence repeats by PCR assays are the defining feature of MSI tumors. Besides the acquisition of new microsatellites, it has been shown that tandem repeats within variable number of tandem repetition (VNTR) regions are increased in gastric cancer [23
]. VNTRs harbor the mucin-type O
-glycosylation sites in mucin proteins and the sole increase of these tandem repeats has been demonstrated to result in the de novo
expression of the TF antigen [24
]. The fact that in average only 30% of the cancer cells were positive supports the hypothesis that the TF expression does not convey a significant growth advantage to cancer cells but arises as a passenger alteration in cells with defective mismatch repair.
In summary, we presented here for the first time strong evidence that TF is a novel, single marker of MSI in gastric cancer. This finding lays the basis for a new line of research and shows great promise to significantly improve the identification of this distinct cancer subtype in the clinical setting.