Whole-exome sequencing reveals the etiology of the rare primary hepatic mucoepidermoid carcinoma

A 64-year-old male was admitted to the Second Affiliated Hospital of Nanchang University on September 16, 2019 with a complaint of repeated abdominal distension over a period of 1 year. He had a previous history of icteric hepatitis and unknown biliary tract surgery 30 years prior and coronary artery stenosis disease with an implanted stent 1 year ago. No abnormality was found in the physical examination. Most preoperative laboratory tests were in the normal range, including serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, total bilirubin, electrolyte, glucose and platelet count and serum alpha fetoprotein level. Hepatitis B surface antigen and hepatitis C antibody were both negative. Abnormal blood test results included C-reactive protein 112.3 mg/l (normal value, < 10 g/l), white blood cell 9.82 × 10⁹/l (normal value, 3.5 × 10⁹/l), red blood cell 3.68 × 10⁹ (normal value, 4.3–5.8 × 10⁹/l), hemoglobin 103 g/l (normal value 130–175 g/l), alkaline phosphatase 483.9 U/L (normal value 45–125 U/L), γ-glutamine dehydrogenase 404.84 U/L (normal value 10–60 U/L), carcinoembryonic antigen (CEA) 10.2 ng/mL (normal value, < 5.0 ng/mL) and carbohydrate antigen 19–9 (CA19–9) 151.2 U/mL (normal value, < 37 U/mL). The abdominal non-contrast computer tomography (CT) scan revealed a low density mass measuring approximately 10 cm in diameter at the left hepatic lobe and intrahepatic bile ducts with multiple stones (white arrow) (Fig. 1a). Enhanced CT scan (arterial phase) revealed a heterogeneous enhanced mass in the left lobe of the liver (Fig. 1b); enhanced CT scan (portal phase) revealed a persistent inhomogeneous enhanced mass and formation of tumor thrombus in the left branch of the portal vein (Fig. 1c) and a persistent inhomogeneous enhanced mass in delayed phase (Fig. 1d). Ultrasonography and magnetic resonance imaging further validated the results of CT. Gastroscopy revealed chronic non-atrophic gastritis. Bone scan and chest CT were performed and excluded metastatic disease. CT scan revealed no solid mass in the head and neck, parotid gland, thyroid gland and pituitary gland (data not shown). The patient underwent resection with left hemihepatectomy and choledocholithotomy and T-tube drainage. The patient was diagnosed with HMEC based on malignant squamous cells and mucus-secreting cells in pathological examination. There was gradually increased jaundice in the postoperative term, and the patient died of hepatic function failure at postoperative 3 months.

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University, and informed consent was obtained from the patient and his family. Parts of tumor tissues and corresponding non-tumor tissues were immediately conserved in a liquid nitrogen tank at − 80 °C. Some tissue samples were fixed in 10% formaldehyde and paraffin-embedded for pathological diagnosis and immunohistochemistry. Exome sequencing and Sanger sequencing were performed in fresh tumor tissue and corresponding non-tumor tissue. The WES data from this study were deposited in NCBI Sequence Read Archive under accession SRA:SRP 266690.

Hematoxylin-eosin staining and immunohistochemistry were performed on tumor samples and corresponding non-tumor samples following standard procedures. Primary antibodies included antibodies against MUC1 (1:200), MUC5AC (1:200), CK7 (1:250), CK19 (1:150), p63 (1:150) and CEA (1:250) (all from Leica, Wetzlar,Germany). All tissue sections were processed using the Lab Vision system (Thermo Scientific, Kalamazoo, MI, USA). The two senior pathology specialists from the Affiliated Hospital of NanChang University (XY Su and LP Xu) who examined the results suggested a diagnosis of HMEC.

FISH was performed on formalin-fixed paraffin-embedded sections of HMEC using a commercially available Dual Color fusion Probe (F.01358, Anbiping Medical Laboratory, Guangzhou, China) following the manufacturer’s instructions. Green fluorescence–labeled CRTC1/MECT1 (19p13) probe (G) and red fluorescence–labeled MAML2 (11q21) probe (R) were used to evaluate the fusion gene. In samples harboring the fusion gene, the green signal and the red signal from probe binding generates a yellow signal (F). The normal signal mode is 2G2R and the typical positive signal mode is 1G1R2F. Histopathology was evaluated by pathologists to determine the tumor and hybridization area. FISH was performed for the CRTC1/MECT1-MAML2 fusion gene and 200 interphase tumor cells were examined in at least two visual fields.

Exome capture was performed using 0.6 μg genomic DNA per sample. Sequencing libraries were generated using the Agilent SureSelect Human All Exon V6 kit (Agilent Technologies, CA, USA). The DNA libraries were sequenced on an Illumina HiSeq platform and 150 bp paired-end reads were generated. The original fluorescence image files were transformed to raw data by base calling and clean data were recorded in FASTQ format. Valid sequencing data were mapped to the reference human genome (UCSC hg19) by Burrows-Wheeler Aligner software [9] and results were stored in BAM format. Samtools mpileup and bcftools were used for variant calling and to identify single-nucleotide variants (SNVs) and InDel [10]. Functional annotation ANNOVAR was performed for annotation for Variant Call Format and detecting variants by 1000 Genome, dbSNP and other databases [11‐13]. Given the significance of exonic variants, gene transcript annotation databases such as Consensus CDS, RefSeq, Ensembl and UCSC were also included to determine amino acid alterations [14]. Using the paired samples of tumor tissue and corresponding non-tumor tissues, somatic SNVs were detected by MuTect [15], somatic InDels by Strelka [16] and somatic CNVs by Control-FREEC [17]. SIFT, Polyphen/Polyphen2 and MutationAssessor were used to predict the impact of SNVs on function [18‐20]; and truncated mutations (nonsense and frameshift) also resulted in high functional impact alterations.

The search and analytic strategy was performed using public data (http://​www.​cbioportal.​org/​) [21, 22]. SNVs were examined in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CHL). We also downloaded SMEC SNV data [23] from published studies to examine the relation between the alterations in the current case with downloaded data. We used an online tool to construct Venn diagrams (https://​bioinfogp.​cnb.​csic.​es/​tools/​venny/​).

Purified DNA was isolated from fresh tumor tissue and corresponding non-tumor tissue from the proband and peripheral blood lymphocytes of four family members using the TIANamp Genomic DNA Kit (Beijing Biotech, China). PCR was performed using the PTC-200PC instrument (BIO-RAD, USA). Primer sequences are shown in Supplementary Table 1. The PCR products were examined using an ABI 3730XL DNA Analyzer (Applied Biosystems, USA).

The mass was an irregular specimen measuring approximately 10 × 7 × 6 cm, revealing no fibrous capsule and tough solid texture; resection from the middle edge showed grayish-white nodules without necrosis. Black pigment stones were also found in the small intrahepatic bile duct (Fig. 1e). The resection edges were free of tumor. There was an absence of cirrhosis in the corresponding non-tumor sample, but massive inflammatory cell infiltration was observed in tumorous and portal areas (Fig. 2a). Hematoxylin and eosin staining revealed that the tumor cells were composed of epidermoid malignant cells, mucous cells and intermediate cells (Fig. 2b). The distribution of epidermoid cells was not nest-like but was surrounded with intercellular bridges, with only a few glandular structures and occasional keratinization. Mucin-producing cells showed mainly abundant clear cytoplasm with eccentric pyknotic nuclei and predominantly showed intracellular mucus and rare extracytoplasmic mucin (Fig. 2c). The heterogeneity of epidermoid cells was obvious, with infiltrated stroma and invasion, but nerve, lymphovascular, blood invasion and necrosis were not apparent. Alcian blue staining revealed mucin in mucin-producing cells (Fig. 2d).

Immunohistochemically, MUC5AC was only positive in the cytoplasm of malignant mucous cells (Fig. 3a). Positive MUC1 was detected at the squamous and mucous tumor cell membrane (Fig. 3b). p63 was diffusely positive in the nuclei of malignant squamoid cells (Fig. 3c). Cytoplasmic CK19 was diffusely positive in malignant squamoid cells and mucous cells (Fig. 3d), and cytoplasmic CK7 was diffuse positive in malignant mucous cells (Fig. 3e). CEA was only focal positive in malignant mucous cells (Fig. 3f).

FISH analysis for the CRTC1/MECT1-MAML2 fusion gene revealed negative results. Two hundred interphase cells were analyzed. The signal patterns were as follows: 2G2R, 7.5%; 3G2R, 40.0%; 3G1R, 5.0%; 4G3R, 10.0%; 4G2R, 5.0%; 4G4R, 7.5%; 2G3R, 5.0%; 3G4R, 5.0%; 2G1R, 5.0%; 3G3R, 7.5%; and 5G3R, 2.5% (Fig. 4).

The sequencing of tumor tissue and corresponding non-tumor tissue in proband provided an average depth on target of 175X and 112X, respectively, and the >10X coverage of the targeted gene regions in tumor tissue and corresponding non-tumor tissue were 94 and 98%, respectively. The total somatic variants included 135 SNVs (53 missenses, 31 synonymous, 3 nonsense and others) and 4 InDels (2 frameshift insertions and 2 frameshift deletions). A total of 252 CNVs (126 gain, 126 loss) were identified in 2591 genes (629 gene gain, 1691 gene loss). GNAS mutation (NM_000516:exon8:c.G602A:p.R201H) was detected in both the tumor tissue and corresponding non-tumor tissue by WES, but the wild-type gene was determined in the corresponding non-tumor tissue by Sanger sequencing. In addition, protein-truncating genetic variants included a frameshift indel mutation in ELF3 (c.909dupC:p.F303fs), and nonsense mutations in DOCK3 (c.C2483A:p.S828X) and KMT2C (c.C1519T:p.Q507X) were detected in tumor samples by Sanger sequencing (Fig. 5D). A Circos map visually summarizes the somatic genomic variations of SNVs, INDEL and CNVs of the HMEC in Supplementary Figure 1.

A total of 1004 significant mutated genes (source: Onco KB) in SNVs from 8 studies containing 1487 HCC patients (Supplementary Fig. 2A) and 654 significant mutated genes (source: Onco KB) in SNVs from 7 studies containing 445 CHL patients were downloaded (Supplementary Fig. 2B) (www.​cbioportal.​org). We also obtained 312 significant mutated genes in SNVs from 18 SMEC patients from the study by Kang et al. Six SNVs (in STAT1, TGFBR1, NOTCH1, KMT2C, ELF3 and GNAS) in the HMEC overlapped with those in primary liver tumors (HCC and CHL), and only an SNV in the CHD3 gene in HMEC overlapped with SMEC (Fig. 5A). Somatic GNAS gene alterations (including missense mutation, amplification and truncating mutation) were detected in 2.1% (9/445) patients with primary hepatobiliary tumors including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma and perihilar cholangiocarcinoma (Fig. 5B). Three patients showed GNAS (p.R201H/C) missense mutation (a putative driver) in exon 8. In addition, Sample ID (W012,T026) were Opisthorchis viverrini–associated cholangiocarcinoma (SRP007970) (Fig. 5C).

The 56 germline variants in 20 Fanconi’s anemia pathway genes included 19 missense variants, 25 synonymous variants and 12 UTR3 and UTR5 in the proband corresponding non-tumor tissue detected by WES (Supplementary Table 2). Five germline homozygous variants and six germline heterozygous variants in six Fanconi’s anemia pathway genes (FANCA, FANCI, FANCW/RFWD3, FANCD1/BRCA2, FANCJ/BRIP1, FAN1) were identified in the proband’s non-tumor tissue by Sanger sequencing (Table 2, Fig. 6A).

There was no family history of Fanconi’s anemia and solid tumor. The proband’s parents died of cardiovascular and cerebrovascular diseases 10 years ago. The older sibling showed hypertension and type 2 diabetes, and the proband’s offspring did not show any disease. The same mutational variants in FANCD1/BRCA2, FANCW/RFWD3 and FAP100/C17orf70 were verified in the pedigree. Heterozygous variants in FANCA (c.G1501A:p.G501S), FANCA (c.A796G:p.T266A) and FANCJ/BRIP1 (c.T2755C:p.S919P) were detected in the proband, but homozygous mutations were shown in the other family members. Heterozygous FAN1 variants were detected in the siblings and proband. Only heterozygous FANCI variants (c.C257T:p.A86V and c.G2225C:p.C742S) were unique in the proband (Table 2, Fig. 6B, C).