Introduction
Gastric cancer (GC) is a heterogenous disease with respect to epidemiology, morphology, and clinical behaviour. Despite declining incidence, GC remains one of the major causes of cancer-related death worldwide [
1]. The incidence of poorly cohesive GC, including signet-ring cell (SRC) cancer, appears to be rising in the Western World [
2,
3]. Several studies investigating the prognostic relevance of SRC histology reported conflicting results [
4‐
8]. In some studies, SRC histology was associated with poor outcome, which was not confirmed in other studies [
4‐
10]. Furthermore, it has been suggested that the relationship between SRC histology and outcome may depend on the disease stage in GC patients [
11‐
17]. The clinical utility of the proportion of SRCs to predict response to preoperative chemo(radio)therapy in GC patients remains a matter of debate [
18‐
24].
A recent expert panel hypothesised that these inconclusive results could be related to inconsistencies in the histological classification of SRC-GC [
25]. Whilst SRC-GC have always been typed as diffuse-type cancers in the Lauren classification [
26], the WHO definition of SRC-GC changed several times between the 1st edition in 1977 [
27] and the 4th edition in 2010 [
28]. Up to the 4th edition [
27,
29,
30], when SRC-GC became a subcategory of poorly cohesive GC, SRC-GC was classified as a separate specific subtype of GC. Furthermore, the definition of the extent of SRCs to qualify as SRC-GC changed over the years from ‘predominant’, to more than 50% SRCs in the 2nd edition WHO [
29] and back to “predominantly” or “exclusively” in the 4th and 5th editions WHO [
28,
31]. In an attempt to achieve a more consistent classification of SRC-GC, a multidisciplinary expert panel recently proposed specific cut-off values for the percentage of SRCs to distinguish bona-fide SRC-GC (more than 90% SRCs) from poorly cohesive GC with SRC component (between 10 and 90% SRCs) and poorly cohesive GC not otherwise specified (less than 10% SRCs) [
25]. However, what remains particularly challenging is the unequivocal definition of what constitutes a SRC based on routine histology as exemplified by the 5 different types of SRCs described in the 3rd edition WHO [
30]. Therefore, there remains an urgent clinical need to identify a specific biomarker for SRCs to standardise SRC-GC classification and establish the clinical importance of SRC-GC.
We hypothesised that (1) SRC containing gastric cancers have a different mucin expression compared to non-SRC gastric cancers, and (2) there is an association between SRC mucin expression, clinicopathological variables, and patient outcome.
The present study consists of two parts: (1) a comprehensive literature search to establish the frequency and clinical importance of mucin stains in SRC containing GC, and (2) a large cohort study in Asian and Caucasian GC where the histological phenotype was classified according to recently published consensus guidelines and the expression of several different mucin stains and its relationship to clinicopathological variables, patient outcome, and ethnicity was investigated.
Discussion
There is an on-going debate whether patients with signet-ring cell (SRC) type gastric or gastro-oesophageal cancer have a different prognosis and response to chemotherapy [
12‐
18] and, therefore, should be treated differently to patients with other gastric cancer (GC) subtypes. The inconsistent results reported in the literature could be related to the fact that the histopathological classification of SRC-GC can be challenging. This is due to the variable morphological appearances of individual SRCs [
54‐
56] which may or may not be recognised as SRCs by some investigators [
54,
54], as well as changing criteria in the WHO classification over the last decades [
27‐
31]. Whilst an expert panel recently proposed cut-offs to enable separating pure SRC-GC (≥ 90% SRCs) from GC with an SRC component, a biomarker to unequivocal identify SRC-GC would be of potential great value to clinicians and patients.
In search of a potential promising biomarker, we decided to focus on mucin-related stains as mucin appeared to be a defining characteristic of SRCs. Our group was the first to conduct a comprehensive literature review into the reported frequency and clinical importance of different mucin stains in SRC-GC. To our surprise, the number of studies describing (immuno)histochemical mucin expression in SRC-GC was very limited and results varied between studies making firm conclusions difficult. However, the existing literature seemed to suggest that mucin is not only present in SRC-GC, but can also be seen in other types of GC [
47,
51,
59,
61,
62]. Furthermore, some SRC-GC appeared to contain no mucin [
34,
35,
39]. Similarly to the well described morphological heterogeneity of GC, we found evidence that combinations of different kinds of mucins can be seen in the same GC [
33,
35‐
40,
45]. Our literature review supports the previous suggestion of an expert panel that inconsistent clinicopathological findings can at least partly be explained by differences in the histological haematoxylin–eosin-based classification of SRC-GC together with different cut-off values used for considering a stain positive. Furthermore, even if the same cut-off value was used, results remained contradictory [
39,
40,
48,
49,
52,
62,
63] which could be related to the use of different primary antibodies [
64]. When comparing reported results from Asian and Caucasian SRC-GC [
34,
39,
41,
49,
58,
62], we saw similar wide ranges of mucin positivity, suggesting that differences might not be simply related to ethnic origin of the cancers. Sampling of the tumours (luminal versus centre versus invasive front) could potentially explain different results in mucin expression as it has been reported by several investigators that intramucosal SRC-GC showed a ‘layered structure’ both morphologically and (immuno) histochemically [
33,
35,
49]. Mucin characteristics of SRC-GC varied depending on tumour size and disease stage [
46,
66], thus results might vary depending on the case selection for the study [
35,
36,
38,
46].
In order to close the gap in the current literature revealed by our comprehensive review, we decided to conduct our own gastric and gastro-oesophageal cohort study into mucin stains. Our study is the largest study to date where all cancers were re-classified in a standardised manner according to WHO classification and recent consensus [
25]. Furthermore, our study is the first study to include both, Asian and Caucasian patients, enabling us to directly compare patient characteristics, histological tumour types, mucin expression, and relationship between mucin expression and patient outcome. Previous studies had either investigated Caucasian cohorts or Asian cohorts.
Our study provides further evidence that there is no (immuno)histochemical mucin stain unique to SRC cancer, since (1) a proportion of SRC containing GC was negative for the (immuno)histochemical mucin stains and (2) a relatively large percentage of GC without SRCs were positive for one or more mucin stains, similarly to what had been reported in the literature [
47,
48,
51,
59‐
62,
65]. However, this is the first study to suggest that the mucin expression might be related to the quantity of SRCs within a given tumour as we saw more frequently mucin expression in poorly cohesive GC containing ≥ 10% SRCs.
When directly comparing Caucasian (LTHT) and Asian (KCCH) GC cohorts, MUC2 and MUC5AC positivity was more frequent in the LTHT cases, whereas ABPAS positivity was more frequent in the KCCH cases. This is the first study to report a relationship between AB positivity and outcome in GC patients. Most interestingly, AB positivity alone or in combination with other mucin expressions was only related to poor outcome in Caucasian GC providing further support for the hypothesis that different outcome in Caucasian and Asian GC patients may be related to an underlying biological difference [
67]. As AB stains acidic mucins which are considered to be present in an intestinal phenotype, our finding would support the described association between (gastro) intestinal mucin phenotype and unfavourable outcome [
38]. Due to relatively low patient numbers in mucin-defined subgroups, we were unable to explore whether the AB expression related outcome difference between cohorts was related to a different disease stage mix.
Based on the literature review and the results of our cohort study, we had to reject our first working hypothesis and conclude that SRC containing GC do not have a different mucin expression compared to non-SRC-GC. Further studies are needed to address this clinical need. On the other hand, results reported in the literature and from our own cohort study confirmed a relationship between SRC mucin expression and patient outcome in the surgery alone setting. Furthermore, our cohort study suggests that irrespective of histological phenotype, the mucin expression is different in Caucasian and Asian GC patient and is associated differently with outcome in different ethnic groups.
We recommend further studies comparing Caucasian with Asian GC to validate our findings and explore underlying molecular mechanisms for difference in mucin expression and outcome. Also, we recommend investigating whether the different mucin expression in SRC containing GC is related to variable treatment response.
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