Clinical and molecular characteristics of mucosal signet-ring cell carcinoma in Helicobacter pylori-uninfected stomach

Background: Gastric cancer develops even in Helicobacter pylori-uninfected patients and its typical histological feature is signet ring cell carcinoma (SRCC) within the mucosal layer. However, the biological characteristics of SRCC remain unclear. We aimed to clarify the pathological and genetic features of SRCC in Helicobacter pylori uninfected patients. Methods: Seventeen Helicobacter pylori-uninfected patients with mucosal SRCCs were enrolled and their clinicopathological characteristics were compared with those of Helicobacter pylori-infected patients with mucosal SRCCs. Seven SRCCs without Helicobacter pylori infection, including two invasive SRCCs, and seven Helicobacter pylori-positive SRCCs were subjected to a genetic analysis using next-generation sequencing. Results: Helicobacter pylori-uninfected patients with mucosal SRCCs revealed male dominancy and a signicantly higher prevalence of smokers among them as compared with the Helicobacter pylori-infected patients with SRCC. A CDH1 mutation (frame shift indel) was detected in one Helicobacter pylori-uninfected cancer not only in the mucosal SRCC but also in the invasive portion. A TP53 mutation was detected in one SRCC without Helicobacter pylori infection. In the control group, ARID1A and TP53 mutations were detected in one SRCC each. The C to A mutation, which is a characteristic smoking-induced mutation, was not found in any of the samples. Conclusions: Some SRCCs in Helicobacter pylori-uninfected patients may have a malignant potential similar to that of SRCCs in Helicobacter pylori-infected patients. Smoking is not a main carcinogenic factor for the development of SRCCs.


Background
It is well recognized that Helicobacter pylori (H. pylori) infection is the main cause of gastric cancer development [1]. In Japan, we previously reported that over 99% of patients with gastric cancer had H. pylori infection [2], and another study reported a similar result [3]. However, gastric cancer may develop even in H. pylori-uninfected patients and its typical histological feature is signet ring cell carcinoma (SRCC) within the mucosal layer [4]. Endoscopically, SRCCs can be easily recognized as whitish depressed or at lesions, and their prevalence and clinical importance have recently been increasing [5].
Previous reports have suggested that SRCCs in H. pylori-uninfected patients show a lower proliferative activity, few extensive spread, and slower progression, compared with SRCCs with H. pylori-infected patients [6]. The features of SRCCs in H. pylori-uninfected patients resemble those in patients with hereditary diffuse gastric cancer (HDGC), which is caused by a germline mutation of CDH1 [7]. In patients with HDGC, a large number of SRCCs can be detected; however, most of them show little tendency to invade into the submucosal layer. Therefore, it is controversial whether SRCCs in H. pylori-uninfected patients can invade into the submucosal layer. In addition, the pathogenesis of SRCCs in H. pylori-uninfected patients remains uncertain. Recently, Horiuchi et al. reported the implication of smoking in the disease pathogenesis of these patients, noting that their ndings should be veri ed by other studies [8].
In the present study, we compared the clinical features of SRCCs between patients with and without H. pylori infection. Moreover, we investigated the genomic characteristics of these lesions to identify their biological characteristics and assess whether smoking may be involved in their pathogenesis.

Patients
We enrolled 19 consecutive patients with SRCC (17 with mucosal cancer and 2 with invasive cancer) without H. pylori infection who were diagnosed in Hiroshima University Hospital from 1998 to 2017. All the patients received endoscopic or surgical resection. The entire tumor was cut into parallel 2-4 mm-thick sections for investigation. Histological evaluation was performed according to the Japanese gastric cancer treatment guidelines [9]. Clinicopathological data was retrospectively reviewed from clinical records. The de nition of H. pylori-negative subjects was judged by our criteria as described in a previous publication [2].
A total of 34 patients with H. pylori-positive SRCC diagnosed in the same period were enrolled as controls. This retrospective study was approved by the institutional review board and the ethics committees of Hiroshima University (No. E-1237) and was performed in accordance with the Helsinki Declaration and its later amendments.

Tissue capture and DNA extraction
In the experiments with next-generation sequencing, we randomly selected seven patients who were habitual smokers among both the H. pylori-negative and -positive SRCCs. Two invasive SRCCs (Case 5 and 6) were included in the H. pylori-uninfected group. In these two cases, the tumors were composed of a mucosal SRCC and a poorly differentiated adenocarcinoma (PDA) component in the deeper portion.
Pathologic tumor tissues were dissected from ten 10-µm-thick slides made from formalin-xed para n embedded (FFPE) specimens, which were depara nized, stained, and dehydrated using the Arcturus® Paradise® PLUS Reagent System (Thermo Fisher Scienti c, Waltham, MA, USA). Dissections were performed using a laser capture microdissection system (LMD 6500, Leica, Wetzlar, Germany), in accordance with the pathogenesis diagnosis. The DNA was extracted from these tissues using the GeneRead DNA FFPE kit (Qiagen, Valencia, CA, USA), and concentrations were determined using a Qubit® 1.0 uorometer (Life Technologies, Carlsbad, CA, NY, USA). The quantity and quality of the FFPE-derived DNA samples were checked by calculating the normalized DNA integrity scores (ΔΔCq) via quantitative polymerase chain reaction (PCR) analysis using the Agilent NGS FFPE QC kit (Agilent, Santa Clara, CA, USA).
Target enrichment and next-generation sequencing DNA extracted from tumors was fragmented into 150-200 bp portions by sonication using an S2 sonicator (6 min, 10% duty, intensity = 5, 200 cycles/burst; Covaris, Woburn, MA, USA) and used for library construction according to the manufacturer's instructions. In all cases, 10 ng of DNA was prepared for sequencing. The exons of ten oncogenes (CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, FBXW7, TGFBR1, RHOA, and MAK2K1) were enriched using the SureSelectXT HS Custom panel (Agilent). The resulting pooled libraries were quality control-checked via the High Sensitivity D1000 ScreenTape system using the 2200 TapeStation instrument (Agilent). Sequencing was performed with paired-end reads via the HiSeq 2500 platform (Illumina, San Diego, CA, USA).

Variant detection
Sequencing reads were aligned to the hg19/GRCh37 reference sequence and analyzed using SureCall 4.0.1 (Agilent). PCR duplicates were removed and low-frequency mutations in template DNA molecules were detected using the molecular barcode system [10]. To identify variants in tumor samples, single sample analysis in SureCall 4.0.1 was used. The called variants were considered germline mutations if they were found in the dbSNP 137 or TogoVar (https://togovar.biosciencedbc.jp) databases. The remaining mutations in cancerous tissues were considered to be candidate cancer-speci c mutations. To reduce the false positive rate, we set the cutoff values for somatic mutation in cancerous tissues as follows: variant score > 0.3; minimum quality for base > 30; variant call quality > 100; allele frequency > 0.1; and number of reads supporting the variant allele > 3 (Supporting le 1).

Statistical analysis
All clinicopathological features were analyzed using the Chi-square test or Fisher's exact test to compare categorical data and Student's t test or Wilcoxon rank-sum test to compare continuous data. A p-value of < 0.05 was considered signi cant. All statistical analyses were performed using JMP® software (SAS International, Cary, NC. USA).

Results
Comparison of clinical features between H. pylori-uninfected and -infected signet ring cell carcinomas First, we compared the clinicopathological features between 17 H. pylori-uninfected and -infected patients with SRCCs located within the mucosal layer. As shown in Table 1, the H. pylori-uninfected patients with mucosal SRCCs revealed male dominancy and a slightly younger age than the H. pylori-infected patients. The SRCCs in the H. pyloriuninfected patients tended to be located in the lower third of the stomach and its tumor size were signi cantly smaller than those in the H. pylori-infected patients. Although no difference was detected in blood type, family history of gastric cancer, or alcohol consumption, the prevalence of smokers was signi cantly higher among the H. pylori-uninfected patients (p = 0.02), although the Brinkman index was not different between the two groups.
Next-generation sequencing of SRCC tissue samples DNA analysis by next-generation sequencing was performed in 16 samples from 14 patients (seven patients from each group) as shown in Table 2. We examined 10 diffuse-type gastric cancer associated genes (CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, FBXW7, TGFBR1, RHOA, and MAK2K1) referring to previous paper [11]. In addition, two DNA samples (one from an SRCC within the mucosal layer and one from a deeper PDA lesion) were extracted from two cases with invasive SRCC without H. pylori infection. In the control group, seven samples were collected from only mucosal SRCC with H. pylori infection. Therefore, in total, nine and seven DNA samples were extracted from the H. pylori-uninfected and -infected patients, respectively.
Finally, a single gene mutation was detected in each of ve samples (Table 3, Figure 1). A CDH1 mutation (frame shift indel) was detected only in one H. pylori-uninfected cancer lesion (case 6) ( Figure 1). This mutation was found not only in the mucosal SRCC but also in the invasive PDA of that patient (Figure 2,3). A TP53 mutation was detected in one SRCC without H. pylori infection (case 4). In the H. pylori-infected control group, mutations in ARID1A (case 11) and TP53 (case 12) were detected in SRCCs (Figure 1). No mutation in CDH1 was found in the control group. Furthermore, the C to A mutation, which is a characteristic smoking-induced mutation, was not found in any of the samples. On the other hand, we did not nd somatic mutation in 7 genes (KRAS, PIK3CA, ERBB3, FBXW7, TGFBR1, RHOA, and MAK2K1) which associated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers [11]. (Figure 1)

Discussion
In the present study, we examined the clinical and genetic characteristics of SRCCs in patients without H. pylori infection. SRCCs in patients without H. pylori infection showed different features compared with those in patients with H. pylori infection. Clinically, the H. pylori-uninfected patients with mucosal SRCCs showed male dominance and the tumor tissue tended to be located in the lower third of the stomach. These observations imply that H. pyloriuninfected SRCCs have distinct biological characteristics and probably have a different carcinogenetic pathway as compared with H. pylori-infected SRCCs.
Particular focus should be placed on the genetic alterations detected in SRCCs without H. pylori infection. Generally, diffuse-type cancers including SRCC are classi ed into the "genomically stable group" according to the four-subtype classi cation [12]. Under these conditions, CDH1 have frequently been detected in SRCCs in patients with HDGC [13]. Moreover, E-cadherin, which is coded by CDH1 gene, regulates signaling pathways leads to increase in cell proliferation, decrease in cell apoptosis followed by gastric cancer development [14]. However, in our study, the prevalence of mutations in CDH1 was extremely low in SRCCs without H. pylori infection. This suggests that the carcinogenic pathway of SRCCs without H. pylori infection differs from that of SRCCs in patients with HDGC. The SRCC lesions found in H. pylori-uninfected patients seem to re ect not only a defect in cell-cell adhesion but also other neoplastic alterations.
Mutations in TP53, which are known as a representative driver mutation in gastric cancer carcinogenesis, were detected in SRCCs without H. pylori infection as well as those with H. pylori infection. This also strongly suggests that SRCCs in H. pylori-uninfected patients may be true neoplastic lesions. Previous reports have demonstrated that SRCC tissue gains an invasive ability after the mutation of TP53 followed by conversion to PDA [15]. Accordingly, at least in a part of H. pylori-uninfected SRCC is a true neoplasm, and clinical features of SRCC without H. pylori infection may be different from that in HDGC. In the present study, we demonstrated the presence of similar genetic alterations between mucosal SRCC and invasive PDA in a H. pylori-uninfected patient, suggesting the progression of SRCC to invasive PDA. Indeed, we have diagnosed two cases with invasive SRCC without H. pylori infection (paper under submission). SRCC without H. pylori infection may have a malignant potential to invade into the submucosal layer and should be treated as a diffuse-type gastric cancer as described in the guidelines for treatment [16].
Notably, it should be emphasized that the prevalence of smokers was higher among the H. pylori-uninfected patients with SRCCs than among the H. pylori-infected patients. This nding is compatible with the previous report by Horiuchi et al. [8]. However, in our study concerning the genetic alterations in SRCC tissue samples, we could not nd any C to A mutations, which are recognized as smoking-induced alterations, despite including samples from some heavy smokers in our analysis [17,18]. Smoking may be a confounding factor as to the pathogenesis of SRCCs without H. pylori infection.
In addition to genetic mutations, gastric carcinogenesis may also be in uenced by epigenomic alterations including gene methylation. Epstein-Barr virus (EB virus) infection, which induces several epigenetic alterations in host genes, may be another carcinogenetic factor besides H. pylori infection [19]. However, it is supposed that H. pylori infection is essential for EB virus-induced carcinogenesis [20]. The pathogenesis of SRCCs without H. pylori infection remains unclear and should be clari ed in future research.
This study has several limitations. The rst limitation is the limited number of patients. To accurately evaluate the prevalence of any given genetic mutation, a large number of cases should be examined. In addition, we examined only ten genes in our cancer panel and we could not cover their complete exon sequences. In clinical practice, SRCCs without H. pylori infection are diagnosed when the lesions measure about 10 mm in diameter; therefore, it is di cult to extract high quality DNA from SRCC tissue. The second limitation is that DNA samples from control lymphocytes were not analyzed. Therefore, we excluded disease-speci c germline mutations and Japanese healthy germline mutations when searching the dbSNP 137 and TogoVar databases.

Conclusions
We have demonstrated the clinical features of patients with SRCCs without H. pylori infection and described some genetic alterations detected in these lesions. Our ndings indicate that SRCCs without H. pylori should be treated as having a malignant potential similar to that for SRCCs occurring in the presence of H. pylori infection.

Declarations
Availability of data and materials The datasets used and analyzed during the current study will be available from the corresponding author on reasonable request. HpU-SRCC: Helicobacter pylori-uninfected signet-ring cell carcinoma, HpP-SRCC: Helicobacter pylori positive (infected) signet-ring cell carcinoma, n.s.: not significant ※ Family History: relative within the 3-degree relationship

Tables
With drinking history, drink alcohol 21.6 g/day or more and drink more than 3 days a week as a drinker. And with smoking history, defined including smoking in present and in the past.   Figure 1 Gastric signet ring cell carcinoma (SRCCs) tissues, paired non-cancerous tissues, from Seven SRCCs without Helicobacter pylori (H. pylori) infection, and seven H. pylori-positive SRCCs were subjected to the targeted panel for 10 genes. The Upper bar-graph shows the number of somatic mutations per sample. The Lower panel shows the type of mutation of each gene. Genes reported SRCC-associated genes in previous paper. First line shows H. pyloriinfect, second line shows smoking status, and from 3th to 12th line shows the type of mutation of each gene. Blue panels shows H. pylori-infected cases, brown panels shows present or current smoker, light-brown panels shows non-smoker, red panels shows frameshift indels or nonsense mutations, and yellow panels shows missence mutations.

Supplementary Files
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