Histopathological Features and Immunophenotype of Primary Gastric Invasive Fibromatosis

Background To investigate the histopathological characteristics, immunophenotype and differential diagnosis of primary gastric invasive bromatosis. Methods The clinical manifestations, histological morphology and immunophenotype of 4 cases of primary gastric invasive bromatosis were observed and related literatures were reviewed. Results Among the 4 patients, 2 were males and 2 were females, aged 28 and 47 years, respectively. The lesions were located in the stomach in 2 cases, the gastric antrum in 1 case and cardia in 1 case. Under the microscope, the tumor was located in the submucosa, growing inltratingly, and inltrating into the gastric wall muscularis, serous membrane and extraserous fatty tissue. Tumor cells were rich in cytoplasm, with unclear cell boundaries, long rod-shaped nuclei, deep chromatin, no atypia, and rare mitotic gures. The tumor tissue was composed of proliferating spindle-shaped broblasts and collagen bers, and the cell morphology was relatively uniform, arranged in parallel bundles or staggered weaves. Tumor tissue invaded and destroyed smooth muscle, blood vessels, nerve tissue and adipose tissue of the gastric wall. Immunophenotype: positive expression of vimentin, β-catenin, SMA positive expression; CKpan, EMA, S-100 protein, desmin, CD99, Bcl-2, ALK, CD34, CD117, DOG1, hormone receptors were all negative; The cell proliferation index ki-67 positive cells were 3–5%.


Background
Invasive bromatosis usually occurs in the broids of the abdominal muscle layer and fascia sheath, so it is also called abdominal wall ligamentoid broids, band tumors, and bromatosis. Because the growth of the tumor is invasive, recurring and locally destructive, it is also known as invasive bromatosis, broid hyperplasia, recurrent abdominal wall broids and abdominal wall broblasts. Invasive bromatosis has no signs of malignancy in cytological morphology, no lymphatic and blood tract metastasis, but it is invasive, recurring, and locally destructive, which is different from benign and malignant tumors [1][2][3][4] . WHO (1994) de nes it as differentiated broblast tumors, whose biological characteristics are between benign broblast tumors and brosarcomas, and are recognized by more and more scholars [5][6][7][8] . Primary invasive bromatosis of the stomach is rare. 3 cases are reported in this article. The clinicopathological features, diagnosis and differential diagnosis are analyzed, and relevant literatures are reviewed. including 2 males and 2 females, aged 28 to 47, with an average of 38.6 years old.
1.2 Methods After surgery, the specimens were xed within 30 minutes, and xed with freshly prepared 10% neutral buffered formalin solution (NBF) for 8-48 hours, and the volume ratio of xative to tissue was 10:1. The tissues of the tumor area were fully collected, and the samples were routinely collected according to the depth of invasion, different colors, and different textures, and stained with HE and immunohistochemical En Vision methods. The primary antibodies vimentin, β-catenin, ALK, EMA, CKpan, SMA, S-100 protein, desmin, CD99, Bcl-2, CD34, CD117, DOG1, ki-67, hormone receptors, and working solutions were all purchased from Fuzhou Maixin Biotechnology Development Co., Ltd., the operation steps strictly follow the kit instructions. Results 2.1 Clinical data There was no obvious cause for upper abdominal pain and discomfort in 4 cases, which was intermittent dull pain, every 2 days or 3 days, the dull pain lasted between 1 hour and 1.5 hours. No discomfort such as acid re ux, heartburn, nausea, vomiting, diarrhea, constipation, etc. No fever, cough, sputum, palpitation, chest tightness, shortness of breath and other discomforts. There were 2 males, one of whom had a history of smoking for 35 years, 15 cigarettes per day, which lasted until hospitalization in 2018. Two females were married at the right age, each had a son, and were healthy. None of the 4 patients had a family history of genetic disease. Admission check: consciousness, good spirits, normal development, and cooperative physical examination. There was no yellowing of skin and mucous membranes, and no swelling of super cial lymph nodes. The thorax was symmetrical and had no deformities, no local bulges and depressions, heart rate 77 to 86 beats per minute, regular rhythm, and normal heart sounds. Specialist examination: at abdomen, tenderness, rebound pain and muscle tension. ECG: Sinus rhythm, normal ECG. Various laboratory tests showed no abnormalities. Two cases of abdominal CT showed a huge soft tissue mass between the gastric body and the gastric wall. The lesion was closely attached to the mucosal layer, and the gastric mucosa was damaged locally. The liver shape and size were normal, the intrahepatic bile ducts were not signi cantly dilated, the gallbladder was normal in size, the pancreas was normal in shape and size, there was no obvious abnormal density in it, and the spleen was normal in size. CT suggested the possibility of gastrointestinal stromal tumors, admitted to surgery with "gastric space occupying" by the outpatient department. Intraoperative frozen rapid pathological diagnosis: (2 cases of gastric body) spindle cell tumor, morphology consistent with invasive bromatosis. (1 case of gastric antrum) the hyperplastic brous tissue showed low-grade intraepithelial tumor changes.
2.2 Pathological examination 2.2.1 Eye view 2 cases are proximal partial gastrectomy specimens, and 2 cases are distal partial gastrectomy specimens. The cut surface of the tumor is gray-white, hard, and the boundary is unclear. No bleeding, necrosis, or cystic change is seen.
2.2.2 Microscopic examination Under the microscope, the tumor is found in the submucosa and shows invasive growth. The tumor tissue invades the gastric mucosa layer, destroying the mucosal layer structure (Figure 1), the mucosal muscle layer mostly disappears, the proper glands are atrophy, hyperplasia, and heterogeneity, forming a disordered shape, the tumor tissue can eventually completely replace the gastric mucosa layer, and the gastric mucosa necrotizes and forms ulcers. The tumor tissue invades the muscular layer of the gastric wall, and the smooth muscle tissue of the muscular layer of the gastric wall is divided into nest-like or disordered pieces of different sizes, especially in the outer muscle layer of the gastric wall, this smooth muscle piece may be pushed by tumor tissue to 10mm away ( Figure   2), indicates that the invasion of the tumor is not only strong, but also has a certain directionality. The smooth muscle fragments destroyed by tumor invasion gradually become smaller, disappear uniformly, and nally are completely replaced by tumor tissue. The tumor tissue invaded the serosal layer to widen the serous membrane, and the tumor tissue divided the fat tissue into irregular pieces of fat. The tumor tissue continued to grow in the adipose tissue outside the serous membrane of the gastric wall, showing a crab-food-like growth ( Figure 3). The directional and invasive destruction of the morphological features of smooth muscle, blood vessels and nerve tissue by tumor tissue is one of the key points in the diagnosis of gastric primary bromatosis ( Figure 4). The tumor tissue is mainly composed of proliferating spindle broblasts and collagen bers, and the morphology of tumor cells is relatively consistent. The arrangement of tumor tissue growth is mainly in bundles (parallel bundles or cross bundles), and can also form a staggered weave-like arrangement. The collagen bers form a wide and long ribbon, sometimes very eye-catching ( Figure 5). The density of cells near the muscularis of the gastric wall is slightly higher, and the density of adipose tissue tumor cells near the serosa is lower, which may be related to the density of the original tissue. There is no necrosis of tumor tissue. In tumor cytology, the tumor cells are long spindle-shaped, with rich and lightly stained cytoplasm, unclear cell boundaries, and elongated spindle-shaped or rod-shaped nuclei with deep staining. The tumor cells have no atypia, with 0 to 3 mitotic gures/50HPF. Ganglion-like myo broblasts can be seen in the tumor tissues, the nucleus is vacuolated, with or without eosinophilic nucleoli, and the cytoplasm is rich and double stained. The accumulation of lymphocytes is often seen in tumor tissues. The main composition is small lymphocytes with no lymph node structure, no germinal center, no envelope structure, and the size is 0.5 to 1.0 mm.

Discussion
Invasive bromatosis or ligament-like bromatosis is the abnormal proliferation of clonal broblasts and/or myo broblasts that occur in deep soft tissues, and has the characteristics of in ltrating growth into surrounding soft tissues and high local recurrence tendency. The etiology of the disease is currently controversial. The literature puts forward three hypotheses [5]: (1) Injury factors, pregnancy, childbirth, abdominal wall muscle ber injury and bleeding, and hematoma formation provide conditions for tumor occurrence; (2) Endocrine disorders, taking estrogen drugs; (3) Chromosomal abnormalities, that is, the Y chromosome corresponding to chromosome 8 / 20 has a triploid. According to the WHO classi cation of soft tissue tumors in 2013, it belongs to intermediate local invasive tumors [1][2][3] . The peak age of onset is between 10 and 40 years old, and there is no obvious gender difference. According to the location of the tumor, it can be divided into three types: abdominal wall bromatosis, extra-abdominal bromatosis, intra-abdominal and mesenteric bromatosis. Extra-abdominal ligamentoid bromatosis mainly affects the skeletal muscle system of the neck, shoulders, and limbs, and a small number of cases involve the intracranial, thoracic cavity, breast, thyroid, liver, etc. [2][3][4] . Primary gastric invasive bromatosis is rare, and the clinical manifestations are upper abdominal discomfort. This study found that the age of onset was 28 to 46 years old, with an average of 37.3 years old. The tumor is located in the submucosa and grows invasively. Key points of histopathological diagnosis: (1) The tumor has no envelope and has the characteristics of strong invasion and destruction of surrounding tissues without causing tissue necrosis; (2) The tumor cell nucleus is elongated, spindle-shaped or rod-shaped, and the cell morphology is relatively consistent, no atypia, mitosis is rare, cell proliferation index is low; (3) The density of tumor cells that invade the smooth muscle tissues of the gastric wall is high, and the density of tumor cells that invade the fat tissues outside the serosal membrane is low; (4) Directional destruction invades smooth muscles and blood vessels and nerve tissue; (5) The tumor tissue is rich in collagen bers, less in ammatory cell in ltration, and there may be lymphocyte aggregation, about 0.5-1.0 mm in size.
Differential diagnosis: (1) In ammatory brous polyps are interstitial hyperplasia composed of spindle cells, small blood vessels and in ammatory cells, especially eosinophils. The age of onset is older, usually 60 to 75 years old, and it is a sessile polypoid mass with an average diameter of 1.5 cm. Histologically, in ammatory brous polyps are composed of loose connective tissue, and are mixed with more in ammatory cells and proliferating thin-walled blood vessels, regional edema or mucus-like background. In ammatory cells are mainly lymphocytes and eosinophils, and sometimes eosinophils are the main component of in ammation, often surrounding blood vessels. Sometimes proliferating mesenchymal cells surround the small and medium-sized blood vessels, forming a concentric structure. The immunophenotype is positive for vimentin and CD34, focally expressing SMA, and negative for βcatenin and ALK [9] . (2) Gastrointestinal stromal tumor (GIST): GIST is the most common mesenchymal tumor of the gastrointestinal tract, which usually occurs in the elderly, with a median age ranging from 60 to 65 years. Gastric GIST can occur in any part of the stomach, from the smallest mural nodules to large complex masses with intracavity and extracavity. The histology of gastric GIST is diverse, most of which are spindle cell type, a few are epithelioid cell type, and mixed type of spindle cell epithelioid cell, and there are a few special morphology, showing sarcomatoid features, accompanied by a large number of nuclear atypia and mitosis, which are pleomorphic cell type. Most gastric GISTs positively express CD117 and DOG1; some positively express CD34 and S1-00 proteins. The KIT or PDGFRA gene mutation can be detected by molecular method [10,11] . (3) Invasive bromatosis in the abdominal cavity involving the stomach: it can be distinguished by the combination of surgical ndings, gross specimen examination and imaging examination. Histologically, invasive bromatosis has the characteristics of directional invasion and destruction of smooth muscle, blood vessels and nerve tissues, which can exclude gastric involvement in abdominal invasive bromatosis [12,13] . (4) Gastric plexiform bromyxoma: it is a rare gastric mesenchymal tumor with unique histological characteristics, which mostly occurs in the antrum of the stomach. Tumors grow in clusters or multiple nodules of varying sizes between the gastric wall, staggered with the smooth muscle of the gastric wall. The tumors are rich in small, thin-walled blood vessels, and the cells are rich in mucin-like or bromycin-like stroma; the tumor cell nuclei are spindleshaped or oval, nucleolus is not obvious, cytoplasm is slightly eosinophilic, and cell atypia is not obvious.
The immunophenotypic spindle cells mainly express α-SMA and muscle-speci c actin (MSA), while βcatenin is negative. Electron microscopy shows that the tumor has myo broblast differentiation [14] . (5) Schwannoma rarely occurs in the stomach, and its frequency is similar to that of gastric leiomyoma. The tumor often occurs in the elderly, and generally forms a mass of 2 cm to 5 cm in the stomach wall. The clinical manifestations, including general features, are similar to those of GlST. The biological behavior of the tumor is benign and recurrence is rare. Tumor cells are composed of spindle-shaped cells, arranged in a cross-bundle shape, and the nuclei can be arranged in a fence shape. Different amount of collagen bers can be seen between the cells. Immunophenotypic tumor cells strongly express S-100 protein diffusely, and some cases express CD34; they do not express CD117, DOG1, ALK, β-catenin, desmin, SMA [15] . (6) In ammatory myo broblastic tumors: mostly occur in children under 10 years of age, mainly composed of proliferating obese spindle-shaped broblasts or myo broblasts, with a large number of in ammatory cell in ltration in the interstitium, mostly mature plasma cells, lymphocytes and eosinophils, a few are neutrophils, and regional lymphocytes aggregate. In addition to the spindle cells, round-shaped histiocyte-like cells can be seen in the lesions. In some cases, some irregular, polygonal or odd-shaped cells can be seen. Eosinophilic or basophilic inclusions can be seen in the nucleus, similar to ganglion cells or R-S cells. The immunophenotype is positive for vimentin and SMA, and desmin and MSA are focally positive. 50% of the cases are positive for ALK, and CD117. DOG1, β-catenin, and S-100 protein are not expressed [16] . (7) Synovial sarcoma: it is a malignant mesenchymal tumor showing epithelioid differentiation to varying degrees, with characteristic chromosomal translocation t(X;18) (p11;q11) and the resulting SS18-SSX gene fusion. Mainly occurring in the limbs of young people, gastric synovial sarcoma is very rare, and 24 cases have been reported in the English literature [17] . A typical synovial sarcoma is composed of cancer-like epithelioid cells and brosarcoma-like spindle cells mixed in different proportions to form a biphasic structure. Gross examination, usually occurs in the gastric body and bottom of the stomach, and occasionally occurs in the gastric antrum or gastroesophageal junction. The tumor is 0.8-16.0 cm in diameter, with multiple or single nodules, uneven surface, and ulcer formation. The section is grayish white, grayish red, sh like, with or without necrosis. Histologically, 22 cases of gastric synovial sarcoma were reported in the literature as monophasic type and 2 cases were biphasic type. Immunohistochemistry shows positive expression of CK and EMA, molecular detection of SYT-SST gene fusion. (8) Myo broma/myo bromatosis: refers to benign tumors formed by muscle-like cells with contractile function arranged around thin-walled blood vessels. The isolated ones are called myo bromatosis, and the multiple ones are myo bromatosis. It can occur from newborns to the elderly, but many are seen at birth and in infants and children under 2 years of age. Men are more common. It mainly occurs in the head and neck, trunk and limbs, as well as skeletal muscle and aponeurosis. 15-20% occurs in internal organs, such as lung, heart, gastrointestinal tract, liver, kidney, pancreas, etc.
Myo broblasts are spindle-shaped, with pale pink cytoplasm, long spindle-shaped nucleus, vacuolated chromatin, with 1 to 2 small nucleoli, and the cell atypia and pleomorphism are not obvious. Round and polygonal cells with slightly larger nuclei can also be seen. The tumor cells are arranged around the irregularly branched perivascular tumor-like thin-walled blood vessels, intersecting with perihemangioma, and the tumor is an image of hemangiopericytoma. Calci cation, hyaline degeneration, necrosis and apoptosis are common. Generally, there are not many lymphocytes and plasma cells. Immunophenotype: positive expression of vimentin and SMA, stronger positive for whole actin HHF35, negative for CK, EMA, ALK, β-catenin, and S-100 protein [18,19] . (9)  CD117, DOG1 [20] . Other tumors that require differential diagnosis include spindle cell hyperplastic nodules, leiomyosarcoma, and epithelioid sarcoma after gastric surgery. Availability of data and materials: The datesets used or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: All of the authors had no any personal, nancial, commercial, or academic con icts of interest separately.  Tumor tissue invades the muscular layer of gastric wall: A. The smooth muscle tissue of the muscular layer of gastric wall is separated into disorganized pieces of different sizes, lumps and nests; B. A smooth muscle mass is visible as the invasion pushes out, up to 10mm away from the musculature.

Figure 3
Page 11/11 Tumor tissue invades the serosal layer; A. Tumor tissue divides adipose tissue into patches; B. Crab-like growth continued to occur between adipose tissues.

Figure 4
Morphological characteristics of directional invasive destruction of smooth muscle, blood vessels, lymphatic vessels and nerve tissues of tumor tissues: A. Neoplastic tissue invades and destroys the smooth muscle tissue, and the smooth muscle bers are separated from the fast slice structure of different size. B. Neoplastic tissue showed invasion and destruction of blood vessels in one direction; C. Neoplastic tissue invades and destroys nerve tissue, leaving only incomplete ganglion cells.

Figure 5
The composition of tumor tissues is mainly composed of proliferating spindle broblasts and collagen bers. The morphology of tumor cells is relatively consistent: A. The arrangement of tumor tissue growth is mainly interlaced bundles. B. Collagen bers form wide and long bands with prominent regions Figure 6 Immunophenotype of gastro bromatosis, En Vision method: a. positive expression of vimentin; Positive expression of -catenin; The number of ki-67 positive cells was 3% ~ 11%.