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Introduction

An inflammatory myofibroblastic tumor (IMT) is a borderline tumor with low malignant potential (1,2), comprised of spindle cells and accompanied by an inflammatory infiltrate of plasma cells, lymphocytes and/or eosinophils (3,4). The lungs, abdominal cavity and gastrointestinal tract are common sites of IMT, but primary hepatic IMT is rare (5). The biological behavior of the tumor remains unclear (6). IMTs are usually benign, but sometimes, malignant biological behavior such as distant metastasis and local recurrence may occur (6,7). The anaplastic lymphoma kinase (ALK) gene, encoding a receptor tyrosine kinase belonging to the insulin receptor superfamily (8), has been found to be rearranged in ~50% of patients with IMT, which results in the overexpression and hyperactivation of the receptor tyrosine kinase (4,9). Therefore, ALK protein has important value in the diagnosis of IMT; for ALK-negative patients, the novel ETS variant transcription factor 6-neurotrophic receptor tyrosine kinase 3 (ETV6-NTRK3) fusion gene, has important reference value for diagnosis (4,10,11).

A biloma is a well-circumscribed intra-abdominal bile accumulation outside the biliary tree (12); it can either be intrahepatic or extrahepatic (12), with an incidence of 0.3–2% (13). The most common cause is choledocholithiasis, and other causes include abdominal trauma and surgery, bile duct tumors, liver infarction, percutaneous catheter drainage, transhepatic cholangiogram and endoscopic retrograde cholangiopancreatography, and fever, nausea, vomiting and epigastralgia are common symptoms caused by biloma (13).

To the best of our knowledge, there are no reported cases of hepatic IMT manifesting with biloma. The present study reports the case of a 45-year-old woman with hepatic IMT negative for ALK upon immunohistochemistry and fluorescence in situ hybridization (FISH) studies, in whom a biloma was identified inside the hepatic IMT.

Case report

In March 2019, a 45-year-old woman presented to Mianyang Hospital of Traditional Chinese Medicine (Mianyang, China) with a solid, well-defined mass in the right upper quadrant of the abdomen. The patient had no abdominal pain, fever, vomiting or jaundice, and no history of hepatitis B or exposure to parasites. Laboratory data showed a white blood cell count, a red blood cell count and a platelet count of 5.17×109/l (reference range, 3.5-9.5×109/l), 4.05×1012/l (reference range, 4.3-5.8×1012/l) and 218×109/l (reference range, 100–300×109/l), respectively, while neutrophil, lymphocyte, eosinophil and basophil levels were 59.9% (reference range, 50–70%), 32.3% (reference range, 20–40%), 1.0% (reference range, 0–7%) and 0.4% (reference range, 0–2%), respectively. Hemoglobin was recorded at 124 g/l (reference range, 120–170 g/l). Alanine aminotransferase, aspartate amino transferase, alkaline phosphatase, glutamyl transpeptidase and lactate dehydrogenase levels were 33 U/l (reference range, 0–50 U/l), 26.5 U/l (reference range, 0–40 U/l), 74.7 U/l (reference range, 45–125 U/l), 17.4 U/l (reference range, 10–60 U/l) and 145.0 U/l (reference range, 120–250 U/l), respectively. Total bilirubin, indirect bilirubin and direct bilirubin levels were 11.50 µmol/l (reference range, 3.42-20.5 µmol/l), 1.30 µmol/l (reference range, 0–6.84 µmol/l) and 10.20 µmol/l (reference range, 0–18 µmol/l), respectively. α-fetoprotein and carcinoembryonic antigen results were 3.21 µg/ml (reference range, 0–20 µg/ml) and 1.30 µg/ml (reference range, 0–5 µg/ml), and carbohydrate antigen 125 (CA125), CA153 and CA19-9 results were 7.50 KU/ml (reference range, 0–35 KU/ml), 10.60 KU/ml (reference range, 0–35 KU/ml) and 7.30 KU/ml (reference range, 0–35 KU/ml), respectively.

Computed tomography of the abdomen confirmed the presence of the lesion, revealing a low-density mass measuring 11.2×8.5×10.5 cm in the left lobe of the liver, with a lower-density mass measuring 8.5×6.1×5.9 cm in the interior of the tumor (Fig. 1A and B). Based on the analysis of patient history, laboratory data and imaging data, the characteristics of the lesion were different from hepatocelluar carcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, liver abscess and hepatic echinococcosis. Furthermore, IQQA-3D (EDDA Technology, Inc.) imaging of the neoplastic area was performed for precise preoperative evaluation (Fig. 2), and the lesion could be easily removed by radical resection. As suspicion of a malignancy remained high, surgical resection of the left hepatic lobe, including the tumor, was undertaken. Intraoperatively, a tumor (12×10×9 cm), with an unclear boundary, incomplete capsule and fish-like texture, was found in the left lateral lobe of the liver, and a biloma, measuring 8×6 cm, was identified inside the tumor (Fig. 1C). The resection margins were clear.

Figure 1. IMT upon abdominal computed tomography and its gross morphology. (A and B) IMT on abdominal computed tomography. (C) Gross morphology of the IMT. The blue arrows indicate the IMT and the black arrows indicate the biloma. IMT, inflammatory myofibroblastic tumor.

Figure 2. IQQA-3D (EDDA Technology, Inc.) imaging of the tumor.

The histopathological review of the sections (4-µm-thick) was performed using hematoxylin and eosin staining. The tumor excision tissue was stored as wax blocks in routine storage at ambient temperature prior to staining. The fixation protocol involved the use of 10% neutral buffered formalin as the fixant, with the duration of fixation being 24 h at room temperature. Following dehydration in a series of ethanol solutions, the tumor excision tissues were paraffin-embedded and sliced into 4-µm-thick sections. Firstly, the sections were deparaffinized in xylene (10 min three times) and rehydrated by serial soaking in 100% ethanol for 10 min, and 95, 85 and 75% ethanol for 5 min each at 25°C. Secondly, sections were stained in 0.1% hematoxylin solution (10 min at 25°C) and differentiated in 1% hydrochloric alcohol, then rinsed with tap water and with distilled water until the nuclei became blue, and dehydrated in 95% ethanol. Thirdly, sections were counterstained in 0.5% eosin solution (3 min at 25°C), and incubated in 95% anhydrous ethanol for 5 min twice and in xylene solution for 10 min at 25°C. Lastly, the sections were sealed with Canada gum. Normal light microscopy was used to image the H&E staining at a magnification of ×200. Spindle cell proliferation with infiltration of chronic inflammatory cells and mucinous degeneration were found (Fig. 3).

Figure 3. Hematoxylin and eosin staining, and immunohistochemistry. (A) Fibroblastic spindle cells and inflammatory infiltrate upon hematoxylin and eosin staining (magnification, ×200). (B) Mucinous degeneration identified by hematoxylin and eosin staining (magnification, ×200). (C) ALK detection by immunohistochemistry, showing a lack of staining (magnification, ×200). (D) Positive vimentin detection by immunohistochemistry (magnification, ×200).

For immunostaining, the streptavidin-peroxidase method was adapted to detect protein expression using paraffin sections. The tumor excision tissue was stored as wax blocks in routine storage at room temperature. The fixation protocol involved the use of 10% neutral buffered formalin as the fixant, with the duration of fixation being 24 h at room temperature. Tissue sections (4-µm-thick) were dried at 60°C for 2.5 h, deparaffinized with two 10-min washes in xylene, and rehydrated in decreasing concentrations of ethanol in distilled water (100, 95, 85 and 75%; 5 min each). Next, sections were soaked in boiling sodium citrate buffer (20140006; Henan Celnovte Biotechnology, Co., Ltd.) for 2.5 min in a microwave oven. When cooled to room temperature, the sections were washed for 5 min in distilled water two times and for 5 min in PBS (20140002; 0.01 M pH 7.2-7.4; Henan Celnovte Biotechnology, Co., Ltd.) one time. Subsequently, sections were treated with 3% hydrogen peroxide at room temperature for 5 min to block endogenous peroxidase activity. After washing for 5 min in PBS two times, the sections were incubated for 60 min at 18–25°C with ALK (rat anti-human) antibody (202011001; clone 5A4; dilution, 1:100; Henan Celnovte Biotechnology, Co., Ltd.) and washed for 5 min in PBS two times, followed by incubation with HRP-labeled goat anti-rat IgG antibody (20170011; dilution, 1:100; Henan Celnovte Biotechnology, Co., Ltd.) for 20 min at 18–25°C, washing with PBS for 5 min (two times), incubation for 3–5 min at 18–25°C with diaminobenzidine (DAB; DAB substrate:DAB buffer=1:20; 20170011; Henan Celnovte Biotechnology, Co., Ltd.), washing for 5 min in distilled water two times, and counterstaining with 5% hematoxylin (20170011; Henan Celnovte Biotechnology, Co., Ltd.) for 5 min. Finally, the sections were stained blue in 1% hydrochloric-acid alcohol at room temperature, dehydrated in increasing concentrations of ethanol (85, 95 and 100%), cleared with xylene, and were sealed with Canada gum.

In order to ensure the accuracy and reliability of the staining conclusion, both positive control and negative control experiments were set up for each section to be detected according to the manufacturer's instructions during the experiment. A blank control was used as a negative control and an anaplastic lymphoma tissue section was used as a positive control. Analysis was performed using an Olympus BX 53 light microscope (magnification, ×200; Olympus Corporation). The aforementioned steps were repeated for CD117 (clone EP10), CD34 (clone QBEnd/10 mouse), discovered on GIST-1 (clone RBT-DOG-1), desmin (clone EP15), smooth muscle actin (clone IA4), S-100 (clone poly), CD21 (clone EP64), pan-cytokeratin (clone AE1/AE3), epithelial membrane antigen (clone aP1.4), CD23 (clone, aR013), vimentin (clone V9) and CD35 (clone RLB25 mouse) (all Henan Celnovte Biotechnology, Co., Ltd.) according to the manufacturer's instructions for each antibody. This revealed negativity for ALK (clone 5A4), CD117 (clone EP10), CD34 (clone QBEnd/10 mouse), discovered on GIST-1 (clone RBT-DOG-1), desmin (clone EP15), smooth muscle actin (clone IA4), S-100 (clone poly), CD21 (clone EP64), pan-cytokeratin (clone AE1/AE3), epithelial membrane antigen (clone aP1.4), CD23 (clone, aR013) and CD35 (clone RLB25 mouse) (all Henan Celnovte Biotechnology, Co., Ltd.), and positive staining for vimentin (clone V9), with 5% Ki-67-positive cells (Fig. 3).

This phenotype is not typical of IMT (11). To confirm the diagnosis, FISH studies were conducted to assess characteristic genetic rearrangements using ALK (Fracture rearrangement probe; cat. no. F.01079; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.), RET (Fracture rearrangement probe; cat. no. F.01104-01; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.), ROS1 (Fracture rearrangement probe; cat. no. F.01086; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.), MDM2 (Fracture rearrangement probe; cat. no. F.01017-01; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.), MGEA5 (Fracture rearrangement probe; cat. no. F.01275-01; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.) and ETV6 break-apart assays (Fusion probe; cat. no. F.01258-01; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.). The tumor excision tissue was stored as wax blocks in routine storage at room temperature. The fixation protocol involved the use of 10% neutral buffered formalin as the fixant, with the duration of fixation being 24 h at room temperature. The tissues were embedded in paraffin. Corresponding H&E sections were reviewed by a pathologist who circled the area of tumor for testing.

ALK FISH was performed using 4-µm-thick FFPE tissue sections. Sections were deparaffinized, rehydrated in 100, 90 and 70% ethanol, washed with 2× saline sodium citrate (SSC) (UltraPure™ 20X SSC; Invitrogen; Thermo Fisher Scientific, Inc.), incubated in pretreatment solution (deionized water; 25 min at 90°C), and digested with pepsin solution (1:10,000; Sigma-Aldrich; Merck KGaA; 4 mg/ml pepsin; 0.02 mol/l HCL) for 6 min at 37°C. Subsequently, sections were washed with 2× SSC (5 min at room temperature), dehydrated in ethanol (70, 90 and 100%), dried at room temperature and then exposed to 10 µl ALK gene probe (Fracture rearrangement probe; cat. no. F.01079; batch number, 202110001; concentration, 60 ng/µl; Guangzhou Anbiping Pharmaceutical Technology Co., Ltd.). Denaturation (5 min at 85°C) and hybridization (10–18 h at 37°C) were performed using the ThermoBrite System (Abbott Pharmaceutical Co. Ltd.). After 24 h, the specimens were treated with 2× SSC (36 ml pure water + 4 ml 20X SSC) for 10 min at 37°C, 2× SSC containing 0.1% Nonidet P-40 (NP 40; Nacalai Tesque Inc.; 36 ml pure water + 4 ml 20X SSC + 40 µl NP-40) for 5 min at 37°C, rehydrated in 70% ethanol (3 min, at room temperature) and dried in the dark. Nuclei were counterstained with DAPI using antifade reagent for 30 min at −20°C (VECTASHIELD Mounting Medium; Vector Laboratories, Inc.). Analysis was performed using a fluorescence microscope (magnification, ×1,000; Axio imager Z1; Carl Zeiss AG) and data analysis was performed using ISIS software (version 5.4.6; MetaSystems). FISH signal was classified under two signal patterns: Normal (two yellow signals) and typical positive signal pattern (one red, one green and one yellow signal), and >100 tumor cells were assessed. The aforementioned steps were repeated for RET, ROS1, MDM2 and MGEA5 genes and ETV6-NTRK3 fusion genes according to the manufacturer's instructions for each gene. These studies found an ETV6 rearrangement, identified as an ETV6-NTRK3 fusion oncogene. ALK, RET, ROS1, MDM2 and MGEA5 were negative (Fig. 4).

Figure 4. Fluorescence in situ hybridization. (A) The anaplastic lymphoma kinase gene was detected using a broken rearrangement probe. The proportion of positive cells was 3% (magnification, ×400). (B) The ETV6 gene was detected using a broken rearrangement probe. The green signal represents the GSP ETV6 (centromere) and the red signal represents the GSP ETV6 (telomere) (magnification, ×1,000). (C) The NTRK3 gene was detected using a broken rearrangement probe. The green signal represents the GSP NTRK3 (telomere) and the red signal represents the GSP NTRK3 (centromere) (magnification, ×1,000). (D) The ETV6-NTRK3 fusion gene was detected using the fusion gene probes. The white arrow shows the typical ETV6-NTRK3 fusion gene, which is demonstrated by the close proximity of the red signal (ETV6 from chromosome 12) with the green signal (NTRK3 from chromosome 15) in multiple tumor cells. ETV6, ETS variant transcription factor 6; NTRK3, neurotrophic receptor tyrosine kinase 3 (magnification, ×1,000). GSP, gene-specific primer.

Finally, a diagnosis of hepatic IMT with biloma was made, based on morphological and immunohistochemical findings, and the characteristic genetic rearrangements. The postoperative course was uneventful, and the patient was discharged at postoperative day 7. The patient was followed up every 3 months without adjuvant treatment. No recurrence of symptoms was noted at the 3-year follow-up and outpatient follow-up every 6 months will still be performed.

Discussion

According to the World Health Organization classification of soft-tissue tumors, IMT is a borderline tumor with low malignant potential; it is a mesenchymal neoplasm comprising myofibroblastic and fibroblastic spindle cells, accompanied by an inflammatory infiltrate of plasma cells, lymphocytes and/or eosinophils (4,5,14). The lung is the most common site of IMT (15). In addition, IMTs originating from female genital organs (4,16), the intra-abdominal region (17,18), the retroperitoneum (19), the ileocecal mesentery (20) and the pancreas (21) have also been reported. However, those located in the liver are even rarer (5).

The ALK gene was first discovered in 1994 in a subtype of anaplastic large cell lymphoma (22), located on chromosome 2p23, encoding ALK (22). Previous studies have confirmed that ALK gene rearrangements are found in nearly one-half of patients with IMT, which leads to ALK overexpression (4,11,23). Therefore, the ALK protein is considered to be an important feature for identifying IMT (4).

For ALK-negative patients, recent studies have identified some new fusion genes, mainly including the ROS1, ETV6, PDGFRβ, RET and NTRK3 genes (10,11,24). A case of ALK-negative uterine IMT harboring the ETV6-NTRK3 fusion gene was reported by Takahashi et al (4). Alassiri et al (11) also found that ETV6-NTRK3 is expressed in a subset of ALK-negative IMTs. These findings are consistent with the characteristic genetic rearrangements of the present patient.

The biological behavior of the tumor is an important factor affecting the prognosis of the patient, as well as an important reference for developing a treatment plan. However, the biological behavior of IMT is unclear. ALK is more commonly expressed in children and is associated with tumor aggressiveness and high recurrence rates (2). By comparison, ALK-negative IMT may have a higher risk of metastasis (2,10).

As is well known, biliary injury is a direct cause of the formation of biloma (25–27). Sakamoto et al (28) found that the incidence of intrahepatic biloma formation in patients with a metastatic liver tumor was higher than that in patients with hepatocellular carcinoma. In the present patient, a biloma, measuring 8×6 cm, was found inside the ALK-negative IMT. We hypothesized that in this case, the formation of the biloma was associated with the lack of expression of the ALK gene and the formation of the ETV6-NTRK3 fusion gene.

To the best of our knowledge, this is the first report of a case of ALK-negative hepatic IMT harboring the ETV6-NTRK3 fusion gene and manifesting with biloma. However, the underlying mechanism of this disease remains unclear. von der Thüsen et al (29) found that the ETV6-NTRK3 fusion gene encodes an activated membrane receptor kinase protein, which can promote cell proliferation and survival through the activation of the Ras-MAP kinase and PI3K-Akt pathways. The ETV6-NTRK3 fusion gene is also associated with secretory carcinoma (30,31). Tang et al (32) reported a case of secretory carcinoma of the breast harboring the ETV6-NTRK3 fusion gene, which showed chemo-resistance to neoadjuvant chemotherapy and multiple distant metastases. Accordingly, we hypothesize that the ALK-negative hepatic IMT with an ETV6-NTRK3 fusion gene in the present case was significantly more likely to invade and damage the biliary tract, which eventually led to the formation of the biloma. As no similar cases have been reported in the past, it remains uncertain as to whether this is a significant observation, and further research will be required to illuminate the mechanism of this disease.

In conclusion, the present case is of particular interest for two reasons. First, it is not a typical case of hepatic IMT owing to the ALK-negativity of the tumor upon immunohistochemistry and FISH, as well as the presence of an ETV6-NTRK3 fusion. Secondly, the report provides the first demonstration of a biloma in an ALK-negative IMT of the liver.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Authors' contributions

KH and JY designed the study and wrote the manuscript. PWZ, JYZ, PZ and KH performed all the experiments. JY, PWZ and KH confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

This study has been approved by The Mianyang Hospital of Traditional Chinese Medicine (Mianyang, China; approval number, 2020LL-12). Written informed consent was obtained.

Patient consent for publication

Written informed consent for publication was obtained.

Competing interests

The authors declare that they have no competing interests.

References