Introduction
Gastric cancer (GC) is histologically a very heterogeneous disease, and this is reflected in the numerous proposed histological classification schemes [
1]. The temporal development of different histological phenotypes in GC remains unclear. Recent studies suggest that Kirsten Rat Sarcoma Viral Oncogene Homolog
(KRAS) activation and downstream signalling can impact on the properties and functions of the tumour microenvironment [
2], and thus may influence histological phenotype. Likely mechanisms of
KRAS activation include
KRAS mutation (
KRASmut) and
KRAS amplification (
KRASamp) [
3].
Mutations in
KRAS have been identified in many human cancers and result in the constitutive activation of
KRAS and the receptor tyrosine kinase (RTK) pathway [
4]. The frequency of
KRASmut is variable across different cancer types, with the highest frequency in pancreatic cancer (90%) followed by colon (34.6%), lung (16.5%) and ovarian (11%) cancer and the lowest frequencies in cervical (6.6%), prostate (5%) and oesophageal cancer (2%) [
5]. In a review of the literature we identified, on average, only 6.5% of GC have a
KRASmut [
6]. In colorectal cancer, routine testing for
KRASmut is now implemented as a predictor of response to anti-epidermal growth factor receptor (EGFR) therapy [
7].
Several studies have demonstrated a relationship between
KRASmut status and histological phenotype in lung and ovarian cancer. In the subgroup of invasive mucinous adenocarcinoma of the lung,
KRAS is mutated in up to 86% of cases [
8]. In ovarian cancer,
KRASmut has been identified in almost all cases with a mucinous histological phenotype [
9]. The relationship between
KRASmut status and histological phenotype in GC remains to be clarified [
6].
The reported frequency of
KRASamp is 1–9% in GC [
10‐
16]. There are no reports of a relationship between
KRAS DNA copy number and histological phenotype in other cancer types and in GC it has not been investigated in a large study. There is increasing recognition of the clinical importance of
KRASamp in GC.
KRASamp is also associated with a worse survival [
3,
10,
12], whereas
KRASmut do not appear to influence survival of GC patients [
17].
Recently, image analysis on lung cancer haematoxylin and eosin (H&E) stained sections using deep learning was predictive of mutation status [
18], thus suggesting that morphological phenotype is reflective of molecular phenotype. Investigating the relationship between
KRAS activation by
KRASmut and/or
KRASamp and histological phenotype may provide some insight into gastric adenoma–carcinoma sequence progression and the origin of histological heterogeneity. Based on the studies in lung and ovarian cancer, we hypothesise that
KRAS activation influences histological phenotype and is associated with a mucinous phenotype in GC. This would suggest that
KRAS activation is an early event in GC, occurring before the phenotype is determined.
The aim of this multicentre GC study was to investigate the relationship of KRAS activation status (KRASmut and/or KRASamp) with the histological phenotype in a large series of GCs from UK, Japan and The Cancer Genome Atlas (TCGA). In addition, the relationship between KRAS status, clinicopathological variables, survival and microsatellite instability status was assessed.
Discussion
This is the largest multicentre study to date to investigate the relationship between
KRAS activation by mutation and/or amplification and histological phenotype in GC. The frequency of
KRASamp (7%) was slightly higher than that of
KRASmut (5%) which is consistent with other GC studies [
10,
11,
37]. The higher frequency of
KRASmut in the TCGA GC cohort compared to the other cohorts could be related to the methodology as TCGA used whole-exome sequencing to test non-hotspot regions, whereas other studies used less-sensitive Sanger sequencing/PCR–RFLP. We found
KRASamp and
KRASmut were exclusive in > 99% of GC, which is consistent with previous reports [
11‐
13,
38].
The relationship between
KRASmut and histological phenotype has not been investigated in great detail and previous studies were limited by small sample sizes and hence lack of statistical power [
6]. In our study, we identified a relationship between
KRASmut and mucinous histological phenotype, which is concordant with higher frequencies of
KRASmut being reported in mucinous lung [
8], ovarian [
9] and colorectal cancer [
39,
40]. However, due to the relatively low frequency of GC with mucinous phenotype and
KRASmut (12%), it would not be feasible to use the presence of a mucinous phenotype as a predictor for the presence of a
KRASmut in GC. The main component of mucinous GCs is extracellular mucin, which consists of high molecular weight glycoproteins regulated by expression of the MUC2, MUC5AC and MUC6 genes in humans [
41]. In mouse models with constitutively activated
KRAS in the stomach, irregular MUC4+ cells were found with abnormal mucins confirmed by Alcian-blue staining [
42]. Interestingly, our study suggests a relationship between
KRASmut and mucinous phenotype, which is characterised by extracellular mucin, but is not related to signet-ring cell type GC, which is characterised by intracellular mucin. Our study confirmed the relationship between
KRASmut and the presence of MSI, which our group and others have described previously in a smaller GC cohort [
43,
44].
The prognostic significance of
KRASmut in GC remains controversial [
6]. In our study, there was no association with the presence of
KRASmut and survival. Interestingly, in lung and colorectal cancer,
KRASmut has been associated with a poor prognosis [
45,
46], whereas in ovarian cancer,
KRASmut has been associated with an improved prognosis [
47].
The relationship between
KRASamp and clinicopathological variables, including histological phenotype in cancer is not well studied. In GC, we found no statistically significant relationship between
KRASamp and histological phenotype, or any other clinicopathological variables. In contrast, others found that the presence of
KRASamp is associated with a poor prognosis in GC [
3,
10,
12]. This difference might be due to case selection and methodology used.
In our study, we used the JGCA scheme for the histological classification of GC and performed a conversion to the Lauren scheme, which is the most widely used histological classification system in Western countries [
22]. Previous studies investigating the relationship between
KRASmut and histological phenotype performed classification according to the Lauren scheme [
6], for which there is no separate category for mucinous GC. The relatively large number of GCs classified as indeterminate according to the Lauren scheme comes from conversion from the JGCA por1 histological phenotype. Direct classification according to the Lauren scheme, would likely result in a higher proportion of GCs classified as either intestinal or diffuse.
In colorectal cancer,
KRASmut is known to be an early event in the progression from normal colonic epithelial cell to adenoma, and finally to carcinoma [
48]. The evidence of sequential development by accumulation of genetic alterations, including
KRASmut, is still controversial in GC [
49‐
51]. We were unable to make any comments regarding the role of
KRAS activation in gastric carcinogenesis in our cohort as we did not investigate precancerous lesions in the current study. However, evidence from mouse models suggest that
KRASmut is one of the key molecular alterations involved in the development of stomach dysplasia [
52] and GC [
53]. Based on the evidence from other cancer types that
KRASmut influence the progression of a mucinous histological phenotype, we therefore speculate based on our results, that
KRASmut in GC is an early event in GC development, whereas
KRASamp is likely to be a late event occurring after the histological phenotype has been established. This would correspond with experiments in mice expressing oncogenic
KRAS in combination with E-cadherin and p53 loss, which resulted in a rapid progression of GC compared to wild type mice [
53].
Our study has some limitations. This is a retrospective study. Histological phenotyping was performed on a single slide. Given the high frequency of intra-tumoural morphological heterogeneity in this study and the previously reported intra-tumoural heterogeneity in
KRASmut status in GC [
54], the sensitivity of some of the techniques used in the current study may not be sufficient to detect
KRAS activation in subclones of tumour cells. As we did not perform microdissection of tumour subregions, we cannot comment on
KRAS status heterogeneity within the same tumour. Furthermore, we used different techniques for DNA extraction,
KRASmut status analysis and MSI analysis in different cohorts included in the current study, each with differing sensitivities [
55,
56].
In summary, we identified a relationship between KRASmut and mucinous histological phenotype in GC. The high level of intratumour morphological heterogeneity could reflect KRASmut heterogeneity, which may explain the failure of anti-EGFR therapy in GC.
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