Identification and characterization of a novel adenomatous polyposis coli mutation in adult pancreatoblastoma

During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/β-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.


INTRODUCTION
Pancreatoblastoma is a rare malignant epithelial neoplasm of the pancreas with multiple lines of differentiation, including acinar differentiation and squamoid nests. Less frequently, this tumor may exhibit endocrine and ductal differentiation and may contain a distinct mesenchymal component [1]. Pancreatoblastoma usually occurs in childhood and is the most prevalent malignant pancreatic tumor in the first decade of life [2]. Exceptionally, it can also be encountered in adults, although such cases are so rare that fewer than fifty cases have been reported to date [3]. No effective therapy has been established, especially in cases with unresectable or metastatic lesions. There have been only a few reports on the molecular features of pancreatoblastoma.

Case Report
Recently, we encountered an adult patient with pancreatoblastoma. Using a next generation sequencer (NGS), loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene was detected in the tumor. In the remaining APC allele, we observed a novel missense mutation, derived from the germline APC gene, which was not reported in the Universal Mutation Database (http://www.umd.be/) or Tohoku Medical Megabank Organization, a Japanese-specific genome cohort (http:// www.megabank.tohoku.ac.jp/). In this report, we characterized the function of the novel APC mutant.

CASE PRESENTATION
A 37-year-old woman visited our hospital complaining of abdominal pain. She had no past or familial history of neoplasms. A computed tomography (CT) scan showed a 5 cm abnormal mass in the pancreas head. She was suspected for pancreatoblastoma by CT-guided biopsy ( Figure 1A) and was sent for a pancreaticoduodenectomy.
The postoperative pathological findings showed squamoid nests, some with keratinization ( Figure 1D). Immunohistological analysis indicated an abnormal nuclear accumulation of β-catenin proteins and the expression of Bcl-10, a pancreatic acinar cell marker, in the tumor tissues ( Figures 1E and 1F). A pathological diagnosis of pancreatoblastoma depends on its acinar differentiation and on the existence of squamoid nests; therefore, this tumor was finally diagnosed as pancreatoblastoma according to these findings.
A CT scan taken three months postoperatively showed metastatic recurrence in the liver ( Figure 1B). She received one course of adriamycin and gemcitabine chemotherapy, with no response. We treated her with cisplatin and S-1 as a second line chemotherapy. Although the tumor temporarily exhibited no change in size, after six cycles of cisplatin and S-1 chemotherapy her liver metastatic pancreatoblastoma increased in size ( Figure  1C). We were unable to achieve any response via salvage treatment, and she ultimately died of pancreatoblastoma progression 13 months after diagnosis.

Analysis of oncodriver mutations in a patient with pancreatoblastoma
We conducted genetic analysis to investigate genetic alterations in the tumor using a cancer panel (OncoDEEP ® , Supplementary Table 1). We identified a previously unreported missense APC mutation of c.5503A > G (p.R1835G). We also confirmed the mutation of the APC gene by Sanger sequencing (Figure 2A) and the expression of APC protein by immunohistochemical staining ( Figure  1G) in tumor tissue samples from the patient. Allele frequency of this mutated allele was 99% in the cancer panel analysis. In addition, we did not find any other alterations of the genes involving the Wnt signal pathway, including CTNNB1. After obtaining written informed consent from the patient, we analyzed her germ line APC gene from her saliva and found a heterogeneous mutation in the 1835 codon of the APC gene ( Figure 2A). These data suggest that a LOH of the APC gene occurred in the tumor, and the products of the APC missense mutation took part in tumorigenesis.
Evaluation of the suppression ability of the Wnt/β-catenin signaling pathway by R1835G mutant APC Immunohistochemical analysis showed an abnormal nuclear accumulation of β-catenin protein ( Figure 1E), indicating activation of the Wnt/β-catenin signaling pathway in the pancreatoblastoma. Therefore, to examine whether R1835G mutant APC affects Wnt/βcatenin signaling, we established cell models expressing APC R1835G mutant protein stably ( Figure 3A). We performed quantitative RT-PCR (q-RT-PCR) to measure the endogenous Wnt/β-catenin transcriptional target, AXIN2, with three genotypes in Caco2 (APC-WT-Caco2, APC-R1835G-Caco2, Cntl-Caco2) and SW480 (APC-WT-SW480, APC-R1835G-SW480, Cntl-SW480). The q-RT-PCR analysis indicated that expression of AXIN2 in APC R1835G was reduced compared to Control (Cntl), but its expression was not downregulated as much as in APC-WT ( Figure 3B and 3C). To examine how APC R1835G affects the Wnt/β-catenin signaling pathway via the TCF/ LEF binding site, we carried out a TOPFlash assay with three genotypes in Caco2 and SW480 ( Figure 3D and 3E). Similar results were also obtained from the TOPFlash assay. These results showed that a R1835G mutation in the APC gene partially inhibits Wnt/β-catenin signaling activity due to the APC mutant directly affecting the β-catenin/TCF transcriptional complex. In addition, we performed an immunofluorescence analysis for β-catenin, comparing the nuclear accumulation of β-catenin among the three genotypes in Caco2 and SW480. As can be seen in Figure 3F and 3G, there was greater accumulation of β-catenin in the nucleus with APC R1835G expressing cell lines than with APC-WT expressing cell lines. It is suggested that the product of APC R1835G has weak ability to bind β-catenin compared with WT.

Evaluation of the suppression ability of the Wnt/β-catenin signal by R1835G mutant APC with external Wnt stimuli
Next, we evaluated whether the R1835G mutant APC had the ability to suppress activated Wnt/β-catenin signaling in HEK293T cells, which originally have a wild-type APC allele. The activation of the Wnt/β-catenin pathway was mimicked by the inhibition of GSK-3β using www.impactjournals.com/oncotarget the pharmacological inhibitor LiCl (lithium chloride). LiCl was added 6 h before the assay. As shown in Figure 4, activation of Wnt/β-catenin signaling was suppressed under the stably overexpressed APC WT. However, the stably overexpressed APC R1835G could not completely suppress activation of Wnt/β-catenin signaling. These results show that the R1835G mutant APC can partially, but not fully, inhibit Wnt/β-catenin signaling activity.

Dose-dependent assessment of suppression ability of the Wnt/β-catenin signaling pathway by R1835G mutant APC
To determine if the suppression ability of Wnt/βcatenin signaling by APC R1835G was dose-dependent, we conducted a TOPFlash assay using ectopically expressed APC R1835G in SW480. Expression of increasing amounts of APC R1835G reduced TOPFlash reporter activity in a dose-dependent manner. We observed enhanced TOPFlash reporter activity of APC R1835G, compared to APC WT ( Figure 5). These results show that R1835G mutant APC attenuate repression of the Wnt/βcatenin signaling pathway activity.

DISCUSSION
This is a case report of pancreatoblastoma in an adult patient. Pancreatoblastoma is a very uncommon neoplasm in adults, and its molecular pathogenesis is largely unknown. We found a novel missense mutation in the APC gene. Although the tumor cells had only novel missense mutation alleles of the APC gene, germ cell lines had wild-type and mutant alleles of APC. Moreover, neoplasms showed abnormal nuclear accumulation of β-catenin protein by immunohistological analysis. Therefore, this case of pancreatoblastoma was likely caused by activation of the Wnt/β-catenin signal by LOH of the APC gene.
According to the cBioPortal for Cancer Genomes (http://www.cbioportal.org/), somatic mutations in the APC and CTNNB1 loci have rarely been identified in pancreatic cancer, but a few CTNNB1 gene mutations have been reported in cases of pancreatoblastoma [4][5][6][7]. Because of the rarity of pancreatoblastoma, the population of APC gene mutations is not known. Only one case has been reported of a germline truncating mutation of the APC gene, a 5-bp deletion at the β-catenin binding site in an individual with familial adenomatous polyposis (FAP) [6]; LOH of the APC gene in pancreatoblastoma was also observed in this case (Table 1). Both pancreatoblastoma and acinar cell carcinomas consistently exhibit acinar differentiation [1], as well as alterations in the genes for Wnt/β-catenin signaling, including inactivating mutations in APC and activating mutations in CTNNB1; however, they lack alterations in the genes commonly mutated in pancreatic duct adenocarcinoma, including KRAS, TP53, P16/CDKN2A, and SMAD4 [8]. We have therefore discussed the activation of Wnt/β-catenin signaling in the pancreas cause not ductal cancer but acinar cell carcinoma, including pancreatoblastoma. This is  consistent with previous reports of alterations in the genes for Wnt/β-catenin signaling, such as APC and CTNNB1, in pancreatoblastoma (Table 1).
APC regulates turnover of cytosolic β-catenin and is the key effector of the canonical Wnt signal pathway. The nucleotide sequence of the APC gene is conserved across species, and APC gene mutations exhibit closely related oncogenesis. The position of the mutation on the APC gene is also important because this gene carries a variety of functional domains, including a heptad repeat, an Arm repeat, a β-catenin binding site (three 15-amino acid repeats and seven 20-amino acid repeats), an Axin binding site (SAMP repeats), a basic region, and an EB1 binding site [9]. The mutation we report here is located between the fourth and fifth 20-amino acid repeat in the β-catenin binding site ( Figure 2B). The mutation, which caused an amino acid to change from hydrophobic arginine to hydrophilic glycine, was located very close to the β-catenin binding domain. Given this, we hypothesized that this mutation may reduce the ability to bind to β-catenin. This hypothesis was supported by the result of the immunofluorescence analysis of β-catenin ( Figures 3F  and 3G).
APC germline mutations are often seen in patients with FAP, with germline truncating mutations frequently observed at the β-catenin binding site, such as in Case 7 in Table 1. In contrast, our patient had no family history of FAP or past history of colon polyposis. This   s t-test), and all assays were performed in triplicate. www.impactjournals.com/oncotarget indicated that the mutation of the APC gene in this case caused pancreatoblastoma, not adenomatous polyposis. Another report described gastric polyposis caused by a missense mutation in the 5′ terminal side of APC [10]; and Ikenoue et al. reported that a truncated mutation at the 3′ terminal side of the APC gene caused attenuated polyposis [11]. In addition, there has been a report of APC missense mutations in cases of hepatoblastoma; however, these cases all featured childhood disease onset, and we were unable to determine whether the patients developed adenomatous polyposis [12].
As described above, in this case the mutation seemed to result in partial loss of biding ability to β-catenin. Differences in the ability to suppress Wnt/β-catenin signaling may account for the phenotypic differences that determine whether adenomatous polyposis occurs. In future studies, we need to investigate the relationship between phenotypes and mutations under in vivo conditions. Comprehensive analysis by NGS gave us a lot of information. On the other hand, we found many variant of uncertain significance (VUS) mutations. There is no established method to confirm if mutations found by clinical sequencing have pathological significance or not. This case report suggests a desperate need for an established evaluation method of VUS mutations in the NGS era.

Mutation analysis by cancer panel and Sanger sequence
The gene analysis of tumor cells was performed using the OncoDEEP Clinical ® (OncoDNA, Belgium), a cancer panel for 409 kinds of genes by NGS. Supplementary Table 1 shows the 409 gene varieties that are detected by OncoDEEP Clinical ® . We submitted five tumor tissue sections with 5 μm thickness and one hematoxylin and eosin staining section to the OncoDNA Company. Almost all exons of the APC and CTNNB1 genes were covered: for APC, exons 1-10, 12, 15, and 16 were 100% covered, exons 13 and 14 were more than 90% covered, and exon 11 was more than 30% covered; for CTNNB1, exons 1-3, 6-9, and 11-15 were 100% covered, and exons 4, 5, and 10 were more than 90% covered.
To confirm the mutation detected by OncoDEEP Clinical ® , we performed Sanger sequencing of the APC gene using a primer, 5′-TCGTCTTCTGCACCCAACAA-3′. For Sanger sequencing of the APC gene from the germline, genomic DNA was extracted from saliva using the Oragene DNA ® (DNA genotek, ON, Canada).

Immunohistochemical staining
Five micron sections of paired neoplastic and normal formalin-fixed, paraffin-embedded tissues were used for immunohistochemical staining with anti-βcatenin (clone β-catenin-1), anti-Bcl10 (clone sc-5273) and anti-APC (clone GTX116009) listed in Supplementary Table 2, according to the manufacturers' instructions. For β-catenin, immunohistochemical labeling was evaluated for the presence of nuclear, cytoplasmic, and membranous β-catenin accumulation in both the neoplasms and the normal surrounding tissues.

Cell lines and culture
Colorectal cancer cell lines Caco-2 and SW480 (APC-null) and embryonic kidney cell lines HEK293T (APC wild type) were cultured according to recommendations of the American Type Culture Collection (ATCC). PLAT-A cells [15] were maintained at 37°C in a 5% CO 2 incubator in DMEM, 10% fetal calf serum (FCS), 1 μg/mL puromycin, 10 μg/mL blasticidin, and penicillin.

Western blotting
Cellular lysates were prepared and the protein concentrations were determined using a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific). Samples with 13 μg and 5 μg of total protein were used for western blots of Caco-2 and SW480, respectively. The samples were separated via electrophoresis on 5%-12% SDSpolyacrylamide gel and then transferred onto a nylon membrane for the western blot analysis. The membrane was probed with the polyclonal rabbit antibodies listed in Supplementary Table 2, anti-APC (clone ab15270), and anti-Vinculin (clone ab73412) as the loading control. Antirabbit IgG.
HRP-linked Antibody (Cell Signaling Technology) was used as the secondary antibody. The protein bands were detected using Pierce ® ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

Immunofluorescence
Caco2 and SW480 cells grown and transfected onto 12-well chambers were fixed for 5 min in 10% methanol at -20° C. The cells were then washed three times with phosphate-buffered saline (PBS) and incubated with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Anti-β-catenin (clone 14/β-catenin) antibody in 1% BSA was then added to the samples, which were incubated overnight at 4° C. After washing three times with PBS, the cells were treated with Goat Anti-Mouse IgG H&L (Alexa Fluor ® 594; Abcam) as the secondary antibody, diluted at 1:200 in 1% BSA for 1 h at room temperature. After washing three more times with PBS, the nuclei were counterstained with ProLong TM Gold antifade reagent with DAPI (Invitrogen). The samples were observed using a BZ-X710 (Keyence).