Genomic profiling of small superficial non-ampullary duodenal epithelial tumors


 Introduction The mechanism underlying carcinogenesis and the genomic features of superficial non-ampullary duodenal epithelial tumors (SNADETs) have not been elucidated in detail. In this study, we examined the genomic features of incipient SNADETs, such as small lesions resected via endoscopic treatment, using next-generation sequencing (NGS). Methods Twenty consecutive patients who underwent endoscopic treatment for SNADETs of less than 20 mm between January 2017 and December 2017 were enrolled. Targeted genomic sequencing was performed through NGS using a 160 cancer-related gene panel. We examined the alteration/mutation frequencies in SNADETs. Results The maximum size of the SNADETs examined in this study was 12 mm in diameter. Five SNADETs were classified as low grade dysplasia (LGD) tumors, while 14 SNADETs were classified as high grade dysplasia tumors. Only one carcinoma-in-situ tumor was detected. We obtained NGS data for 16 samples. APC alterations were detected in 81% of samples (13/16). KRAS, BRAF, and TP53 alterations were detected in 25% (4/16), 18.8% (3/16), and 6.3% (1/16) of cases, respectively. Conclusions We detected APC alterations in most small SNADETs resected via endoscopic treatment, from LGD to carcinoma samples. Even in SNADETs classified as small LGD, KRAS and BRAF alterations were present in a few samples.


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Introduction Superficial non-ampullary duodenal epithelial tumors (SNADETs) are defined as adenomas and superficial adenocarcinomas, including carcinoma-in-situ (CIS) and submucosal invasive cancer of the non-ampullary duodenal area [1]. Duodenal epithelial tumors are extremely rare, with a reported prevalence of 0.4% in patients undergoing esophagogastroduodenoscopy [2]. However, the detection rate of duodenal carcinoma has been increasing owing to the widespread use of endoscopy [1,3].
According to the Vienna classification for gastrointestinal tumors [4], adenomas of the gastrointestinal tract can be categorized as low-grade dysplasia (LGD; category 3) and high-grade dysplasia (HGD; category 4.1). In this classification, the recommendation for HGD or carcinoma is local endoscopic or surgical resection, and the recommendation for LGD is endoscopic resection or follow-up. Tsuji et al. proposed an algorithm for the treatment of SNADETs [5]. Recently, some diagnostic methods based on magnified endoscopy with narrow-band imaging (NBI) or endocytoscopy have been reported [6,7].
In addition, the number of resected SNADETs is increasing owing to improvements in endoscopic treatment [1]. Endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) are the main endoscopic approaches to treating SNADETs.
Therefore, our understanding of the clinical and pathological features of SNADETs is improving [9,10]. However, relationships among the genomic profile and prognosis of SNADETs have not been clarified.
In colorectal cancer (CRC), the adenoma-carcinoma sequence describes the process of carcinogenesis [11]. APC plays a principal role in CRC development as a tumor suppressor gene. Extensive studies of associations between gene alterations in key driver genes and CRC metastasis [12] have demonstrated the significant roles of alterations in KRAS, Tp53, SMAD4, and BRAF. Similar mechanisms to those in CRC, such as the adenoma-carcinoma sequence, may contribute to the pathogenesis of duodenal adenocarcinoma [13]. Genomic analyses of duodenal tumors have reported APC, KRAS, and BRAF alterations [14,15]. However, the mechanism underlying carcinogenesis and the genomic features of SNADETs have not been elucidated in detail.
In this study, we examined the genomic features of incipient SNADETs, such as small lesions resected by endoscopic treatment, using next-generation sequencing (NGS).

APC alterations in most SNADETs from
LGD to carcinoma samples.

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In SNADETs classified as small LGD, KRAS and BRAF alterations were present in some samples.

Subjects and clinicopathological properties of 20 SNADETs
This study included 20 consecutive SNADETs resected by endoscopic treatment ( Table   1). The maximum size of the tumors was 12 mm in diameter. The endoscopic procedures employed were CSP (11 lesions), EMR (8 lesions), and ESD (1 lesion). The case in which ESD was performed had severe submucosal fibrosis because of biopsy. Therefore, we abandoned EMR and chose ESD for tumor resection. Most lesions (85%) were located in the second part of the duodenum. Phenotypic analysis showed no gastric-type lesions.

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We observed a high frequency of APC alterations in SNADETs (i.e., 81%). Additionally, there were no significant differences between the rates of APC alterations in the LGD group (80%) and the HGD/CIS group (81.9%). Kojima et al. reported an APC alteration frequency of 54.5% in duodenal adenoma [15]. APC plays a critical role in CRC development as a tumor suppressor gene, and its gene product inhibits Wnt/β-catenin signaling [19]. Based on a gene set enrichment analysis, Sakaguchi et al. [14] found a strong association between expression profiles in duodenal adenoma/adenocarcinoma and colorectal adenoma after Cre-lox APC gene knockout. These findings suggest that upregulation of the Wnt/β-catenin pathway is a major factor in the initial stages of duodenal adenoma/adenocarcinoma carcinogenesis. Our results further support the key role of APC in duodenal adenoma/adenocarcinomas.
In CRC, BRAF and KRAS alterations typically arise at the adenoma stage of the adenoma-carcinoma sequence [20,21], following an initial APC alteration. KRAS and BRAF encode proteins belonging to the Ras-Raf-MEK-ERK signaling pathway. The activation of this pathway is considered a molecular switch, leading to cell growth and proliferation [22]. Alterations in KRAS and BRAF are associated with a risk of developing advanced neoplasia [23] and contribute substantially to CRC metastasis [12]. In the present study, KRAS, BRAF, and TP53 alterations were detected in 25%, 18.8%, and 6.3% p. 11 of patients, respectively. Surprisingly, we detected KRAS alterations in 40% (2/5) and BRAF alterations in 20% (1/5) of LGD lesions. These findings are consistent with a previous study showing that one in five LGD cases (20%) harbor a KRAS alteration [15].
It has been reported that, even in LGD, large SNADETs ≥20 mm in diameter have a high risk of progression to adenocarcinoma [24]. There were no histological differences between LGD tumors with KRAS or BRAF alterations and those without alterations within wild-type sequences.
TP53 is a key driver gene in CRC progression and is frequently detected in small bowel advanced adenocarcinoma [25]. In this study, one CIS case had a TP53 alteration. These results support the hypothesis that the accumulation of genetic alterations after an initial APC might cause progression from adenoma to carcinoma in SNADETs. Considering our results and those of previous reports [14], SNADET progresses according to an adenomacarcinoma sequence, similar to colorectal tumors. Additionally, more than half of the LGD SNADETs (60%; 3/5) already had KRAS or BRAF alterations, which might cause progression to HGD or carcinoma.
This study had several limitations. It included a relatively limited number of samples and did not include SM invasive cancer samples. Additionally, we performed genome sequencing analysis using the Human Comprehensive Cancer Panel (Qiagen), which p. 12 included 160 cancer-related genes. Therefore, we could not analyze other gene alterations and epigenomic changes in SNADETs. These limitations should be considered when interpreting the study results.

Conclusion
In the incipient SNADETs such as small lesions resected by endoscopic treatment, we detected APC alterations in most SNADETs from LGD to carcinoma samples. Even in SNADETs classified as small LGD (<12 mm in diameter), KRAS and BRAF alterations were present in few samples.

Subjects and Samples
Twenty consecutive patients (20 samples

Specimen handling
All resected specimens were routinely fixed in 10% buffered formalin for 24 hours at room temperature. Then, the specimens were serially sliced at a width of approximately 2 mm and embedded in paraffin following routine methods. All sections were cut to a thickness of 3 μm and stained with hematoxylin and eosin (HE) for microscopic examination. Paired peripheral blood samples were collected from each patient and stored at -80°C.

Clinicopathological assessment
Clinicopathological findings were reviewed, including age, sex, tumor location, tumor color, tumor size, tumor macroscopic type, resection method, histological type, and phenotype of the resected specimen. Macroscopic typing of SNADETs was based on the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma [16]. According p. 14 to endoscopic features, the samples were classified into the elevated (0-I), superficial elevated (0-IIa) or superficial shallow or depressed types (0-IIc). Mixed patterns were diagnosed when more than one component was observed. Histological evaluations were performed by two expert pathologists (SN and YM) who were blinded to the genomic analysis, clinical information, and endoscopic diagnosis. Histopathological diagnosis was based on the revised Vienna classification [4]. Adenomas were subclassified into lowgrade (equivalent to adenomas with mild to moderate atypia) and high-grade (equivalent to adenomas with severe atypia) according to their degrees of structural and/or cytological atypia. CIS showed obvious structural atypia and nuclear atypia. Representative examples of these adenomas and CIS are shown in Figures 1, 2, and 3.

Immunohistochemistry
Immunohistochemical staining was performed using the dextran polymer-peroxidase-

Genomic DNA extraction from tumor tissues and blood cells
Each resected specimen was sectioned into 5 slices (8 μm thick per slice), and macroscopic trimming was performed to obtain as many cancer cells as possible for more than 50% tumor cellularity. Genomic DNA was extracted from formalin-fixed paraffinembedded (FFPE) tissue samples using a GeneRead DNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was extracted from the blood samples using a Genomic DNA Extraction Kit (Katayama Chemical, Osaka, Japan). The concentration and purity of genomic DNA samples were quantified using a NanoDrop system (Life Technologies, Carlsbad, CA, USA) and Qubit dsDNA HS Assay Kit (Life Technologies) designed to be accurate for sample concentrations of p. 16 10-100 ng/mL. Genomic DNAs from the FFPE tissue and blood samples were stored at -80°C until analysis.

Library construction and next-generation sequencing
Multiplex PCR was performed using a GeneReadDNAseq Panel PCR Kit V2 (Qiagen) and Human Comprehensive Cancer Panel (Qiagen), which included 160 cancer-related genes. Finally, an optimized library was constructed using a Gene Read DNA Library I Core Kit (Qiagen). The library was analyzed using an Agilent DNA 1000 Kit Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Library preparation was achieved within 2 working days. The enriched libraries were sequenced to obtain paired-end reads (2 × 150 bp) using the MiSeq NGS platform (Illumina, San Diego, CA, USA), resulting in a mean depth of >500×. The sequencing data were analyzed using an original bioinformatics pipeline, GenomeJack, tuned for clinical sequence examination, "CLUHRC" (Mitsubishi Space Software Co., Ltd., Tokyo, Japan) [18].

Statistical methods
The results were analyzed using Prism version 6 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as means ± standard errors of the mean. Parameters were p. 17 compared between two groups by Fisher's exact test or Student's t-test. Differences were considered statistically significant when p < 0.05.      The 16 samples analyzed by next-generation sequence were divided into two groups (5 LGD and 11 HGD/CIS). There were no significant differences between the alteration frequencies of APC, KRAS, BRAF, and TP53 in the LGD and HGD/CIS groups.

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Parameters were compared between two groups using Fisher's exact test. Differences were considered statistically significant if p < 0.05.