Introduction

Gastric dysplasia is a pre-malignant lesion that can progress to gastric cancer (GC), however the factors that influence its progression is still poorly understood. Here, we aim to identify molecular factors that promote the development of GC from gastric dysplasia.

Aims & Methods

We performed whole exome sequencing on 110 gastric dysplasia or GC samples from 82 patients, with an average exome coverage of 154X including the germline normal comparison. The samples were obtained from 2017 to 2019 and included (a) dysplastic samples from 34 patients without GC (b) GC samples from 20 patients without dysplasia (c) concurrent dysplasia and GC samples from 28 patients.

Results

Classification based on TCGA GC subtypes showed CIN (chromosomal unstable) GC to be more prevalent in GC without concurrent dysplasia (60% vs 21.4%; Fisher test p-value 0.030) whereas MSI (microsatellite unstable) GC tend to be concurrent with dysplasia (21.4% vs 5.0%; Fisher test p-value 0.21), suggesting acquisition of copy number alterations to be associated with rapid progression into GC. Consistent with this hypothesis, we also identified a significant increase in copy number alterations in GC samples compared to dysplasia (Wilcoxon test; p=5.1x10^-3), while somatic mutation rate was more modestly increased (Wilcoxon test; p=0.12). CIN samples were enriched with GC samples, TP53 mutations, 17q12 (ERBB2) amplification and 9p21 (CDKN2A) deletion whereas non-CIN samples showed high APC mutations, 5q23 (APC) deletion and is associated with gastric dysplasia.

Conclusion

The molecular data presented in this study is the most comprehensive to date for gastric dysplasia and early GC. Our results suggest copy number alterations may be the key driver of GC progression in dysplastic samples and molecular profiling may be used to identify patients at higher risk of developing GC.

Introduction

The contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized (1). During GC progression striking changes in the lymphatic vessels (LVs) occur and lymphatic metastasis is the commonest feature of GC spread, representing the most significant prognostic factor determining the clinical outcomes (2). Recent evidence supporting a functional link among EMILIN-1, an Extra Cellular Matrix component (ECM), tumour growth and lymph-angiogenesis has been provided. EMILIN-1 represents a key structural element in the maintenance and function of LVs and displays anti-proliferative effects through the interaction with α4β1 integrin, as demonstrated in skin and colon cancer (3,4,5). Interestingly, α4 integrin is transcriptionally repressed in GC, but not in the normal gastric epithelium (7), suggesting a crucial mechanism exerted by the integrin/EMILIN1 recognition also in this context.

Aims & Methods

The aim of this study is to analyze the possible role of EMILIN-1 as a molecular regulator in in the context of GC development and progression.
We performed Immunofluorescence staining of human gastric biopsy samples from normal and neoplastic tissues collected during the endoscopy procedures. Biopsies from pre-malignant lesions were also included. For in vivo experiments, we used the syngeneic YTN16 gastric cancer cell line (7) which was injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced in mice using N-methyl-N-nitrosourea (MNU). GES-1 (normal stomach mucosa), and several GC cell lines representative of different GC stages and histotypes (i.e. AGS, Hs746.T, NCI-N87 and KATO-III) were employed for in vitro experiments.

Results

EMILIN-1 is diffusely deposited in normal gastric mucosa and is significantly decreased in malignant and pre-neoplastic samples; this finding correlates with the presence of aberrant LVs. EMILIN-1 mutant mice (knock out and E955A knock in) developed tumors earlier compared to WT animals: 20 days post-injection of YTN16 cells, tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities.
To unveil the main mechanisms responsible for EMILIN-1 reduction we treated in vitro fibroblasts and LECs (the major cellular sources of EMILIN1 in the gastric microenvironment) with conditioned media derived from GC cells lines; dramatic effects on EMILIN1 expression were observed, both at the mRNA and protein levels. Small-RNAseq analysis on culture media allowed us to identify miR125a-5p as a possible molecular regulator of EMILIN-1 levels in this context.

Conclusion

This study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

References

1. Rojas A, Araya P, Gonzalez I, Morales E. Gastric Tumor Microenvironment. Adv Exp Med Biol. 2020;1226:23–35.8
2. Deng J-Y, Liang H. Clinical significance of lymph node metastasis in gastric cancer. World J Gastroenterol. 2014;20:3967–75.
3. Capuano A, Pivetta E, Sartori G, Bosisio G, Favero A, Cover E, et al.Abrogation of EMILIN1-β1 integrin interaction promotes experimental colitis and colon carcinogenesis.Matrix Biol J Int Soc Matrix Biol. 2019;83:97–115.
4. Capuano A, Pivetta E, Baldissera F, Bosisio G, Wassermann B, Bucciotti F, et al.Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis. Matrix Biol J Int Soc Matrix Biol. 2019;81:34–49.
5. Danussi C, Petrucco A, Wassermann B, Pivetta E, Modica TM, Del Bel Belluz L, et al.EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation. JCell Biol. 2011;195:131–45.
6. Park J, Song S-H, Kim TY, Choi M-C, Jong H-S, Kim T-Y, et al. Aberrant methylation of integrin alpha4 gene in human gastric cancer cells.Oncogene. 2004;23:3474–80.
7. Yamamoto M, Nomura S, Hosoi A, Nagaoka K, Iino T, Yasuda T et al. Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth. Cancer Sci. 2018 May;109(5):1480-1492.

Introduction

As curative criteria of undifferentiated-type gastric cancer (UD-GC) by endoscopic submucosal dissection (ESD), it is defined lesions fulfill the pathological size ≤ 20 mm, pT1a, absence of ulcerative findings (UL-0), absence of lympho-vascular invasion (LVI-0) and R0 resection in Japanese gastric cancer treatment guidelines. However, additional gastrectomy is needed for other UD-GCs. Meanwhile, it is reported that pathological size of ≤ 40 mm, pT1a, purely UD-GC (without differentiated-type [D-type] components) patients with UL-0 and LVI-0 did not show lymph node metastasis (LNM). Additionally, it is reported that no LNM occurred in the patients with pathological size > 20 mm, pT1a, UD-GC, UL0, LVI-0, and retained double-layer structure (as distribution of cancers in mucosa, the upper layer primarily comprises intracytoplasmic abundant mucin with an eccentric nucleus, and the lower layer primarily comprises poor intracytoplasmic mucin and acidophilic cytoplasm). However, all of these studies were single-center retrospective studies with a small sample size of surgical cases, and there have been no multi-institutional studies with central pathological review (CPR).

Aims & Methods

This was a multicenter retrospective study from 45 Japanese institutions to determine the proportion of LNM, and clarify the clinicopathological characteristics of the lesions without LNM (as no metastasis of lymph nodes in surgical specimens). We included patients pathologically diagnosed with purely UD-GC, lesion size 21-40 mm, pT1a, UL0, LVI-0 by initial gastrectomy or gastrectomy after ESD at each institution. Patient information was entered into an electronic case report form via electronic data capture. In addition, seven unstained slides, including the deepest invasive area of each lesion, were collected from each institution, and hematoxylin-eosin staining and immunostaining for MUC5AC and MUC6 were performed. As a CPR, two pathologists assessed whether UL-0, pT1a, and absence of minor tubular components (as a small amount of tubular components) were observed. UL-1, pT1b, D-type dominance or unevaluable specimens were excluded. Further, whether the lesion involved the entire mucosal layer and whether a double-layer structure was present. The final decision on the lesions with discrepancies was made through discussion with another member of the CPR via web meeting.

Results

Of the 652 patients enrolled, 127 were excluded due to UL-1, pT1b, D-type dominance or unevaluable specimens by the CPR. In 525 eligible patients (initial gastrectomy; 444, gastrectomy after ESD; 81), 10 patients had LNM (1.8%, 95% Confidence Interval [CI] 0.9-3.5%). Comparing 10 patients in the LNM group with 515 patients in the non-LNM group, there were no significant differences in the pre-treatment endoscopic findings. Meanwhile, the results of the CPR revealed that the absence of minor tubular components is more frequent in the non-LNM group than that in the LNM group (365/515, 70.9% vs 3/10, 30%, p=0.011). When stratified by the results of the CPR, patients with preserved double-layer structure did not have LNM (0/41, 95%CI 0-8.6%). Meanwhile, patients without minor tubular components had 0.8% as proportion of LNM, and upper of 95%CI was low among each population (3/365, 95%CI 0.2-2.4%).

Conclusion

There were 1.8% of LNM in all eligible patients. In contrast, as purely UD-GC, lesion size 21-40 mm, pT1a, UL0, LVI-0 without minor tubular components had a low proportion of LNM, they are promising candidates for the expansion as the curative criteria of ESD.