Nanopore DNA Sequencing Detected Chromothripsis-Induced PAFAH1B1::USP6 Rearrangement in Periosteal Solid Aneurysmal Bone Cyst Initially Diagnosed as Osteosarcoma.

An aneurysmal bone cyst (ABC) is a benign bone neoplasm that typically occurs during the first and second decades of life. ABC usually presents as a rapidly growing intramedullary expansile mass with multiple blood-filled cysts in the metaphysis of the long tubular bones. Here, we report a case of a periosteal solid ABC that was initially diagnosed as a high-grade surface osteosarcoma. A 10-year-old male was referred to our hospital for swelling and tenderness of the left upper arm. Radiography revealed periosteal mass without fluid-fluid levels. On performing open biopsy, the tumor showed hypercellular proliferation of uniform spindle to epithelioid cells with brisk mitotic activity (up to 12/2 mm2) and lace-like osteoid formation, which was diagnosed as a high-grade surface osteosarcoma. After one course of chemotherapy using adriamycin and cisplatin, peripheral sclerosis was conspicuous, which led to pathological review and revision of diagnosis as "possibly osteoblastoma." The patient was disease-free for 4 years after marginal resection and curettage. Retrospective nanopore DNA sequencing unexpectedly detected a PAFAH1B1::USP6 rearrangement. The fusion gene was further validated using reverse transcription-polymerase chain reaction and the diagnosis was revised to ABC. Chromothripsis involving chromosome 17 has also been identified. Methylation analysis classified the present tumor as an ABC or non-ossifying fibroma using t-distributed stochastic neighbor embedding and unsupervised hierarchical clustering. This case report highlights the utility of nanopore DNA sequencing for soft tissue and bone tumor diagnosis.

Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

Immunohistochemical assessment of growth factor signaling molecules: MAPK, Akt, and STAT3 pathways in oral epithelial precursor lesions and squamous cell carcinoma.

Several growth factors and their receptors, such as epidermal growth factor receptor, have been studied as prognostic biomarkers for many epithelial malignancies. The signal transduction cascade of those receptors includes RAS/RAF/ERK, PI3K/Akt/mTOR, and STAT3 pathways. The aim of this study was to investigate the expression levels of several key proteins of those pathways in patients with oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), and to correlate the expressions of these proteins with clinicopathologic features and prognosis. Fifteen leukoplakia (LP), 15 low-grade epithelial dysplasia, 15 high-grade epithelial dysplasia (HD), and 132 OSCC specimens were immunohistochemically examined for KRAS, HRAS, NRAS, BRAF, pERK1/2, pAkt, pmTOR, and pSTAT3 expression. Immunoreactivity for these molecules predominantly occurred in regions OEPL basal to prickle layers and in most OSCC cells. KRAS and NRAS expression was significantly lower in OSCC than in OEPLs, while pAkt and pmTOR showed higher expression in OSCC than in OEPLs. pERK1/2 expression was significantly higher in HD than in LP. In OSCC, KRAS and NRAS immunoreactivity was significantly higher in advanced age and male gender. In addition, higher immunoreactivity was shown in pERK1/2 in female gender and advanced TNM stage, pAkt in advanced T classification and cases without postoperative metastasis, pmTOR in advanced mode of invasion, and pSTAT3 in invasion depth. Correlations between these markers and clinicopathological variables were also noted. MAPK, Akt, and STAT3 pathways might play diverse roles in oral carcinogenesis.

Immunohistochemical assessment of Eph/ephrin expression in oral squamous cell carcinoma and precursor lesions.

To evaluate erythropoietin-producing hepatocellular carcinoma (Eph)/Eph receptor-interaction protein (ephrin) expression in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPLs), EphA2, EphB4, and ephrinB2 were examined and compared with microvessel density (MVD) and lymphatic vessel density (LVD). Samples from 73 OSCC and 43 OEPLs patients were immunohistochemically analyzed with antibodies against EphA2, EphB4, ephrinB2, CD34, and D2-40. Results were compared with clinicopathological findings. Immunohistochemical reactivity for EphA2, EphB4, and ephrinB2 was detected in epithelial cells and some stromal vascular cells in OEPLs and OSCC, proportionately with the level of malignancy. The number of blood vessel endothelial cells stained with CD34 and lymphatic vessel endothelial cells stained with D2-40 was increased in OEPLs and OSCC. In OSCC, ephrinB2 and EphB4 exhibited significant correlation with recurrence and invasion depth, respectively. MVD was significantly lower in slight lymphocytic reaction than in prominent stromal reaction. Association was found between LVD and T classification, postoperative metastasis, survival, mode of invasion, and invasion depth. Expression of EphA2, EphB4, ephrinB2, MVD, and LVD might be associated with malignant potential of the oral epithelium. Angiogenesis and lymphangiogenesis appear to be related to progression of potentially malignant oral lesions.

Immunohistochemical assessment of chromatin licensing and DNA replication factor 1, geminin, and γ-H2A.X in oral epithelial precursor lesions and squamous cell carcinoma.

BACKGROUND: Carcinogenesis occurs when the cell cycle is compromised. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X are expressed in cells in G1 phase, S/G2 /M phases, and apoptosis, respectively, and these three markers may be useful for histological evaluation of malignant lesions. Here, we aimed to identify cell cycle phases and apoptosis using immunohistochemistry in oral epithelial precursor lesions and oral squamous cell carcinoma. METHODS: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were immunohistochemically examined in tissue specimens from 55 patients with oral epithelial precursor lesions and 50 patients with oral squamous cell carcinoma. Associations of clinicopathological variables with marker expression were assessed. RESULTS: Chromatin licensing and DNA replication factor 1 was expressed in the prickle cell layer of oral epithelial precursor lesions and many carcinoma cells of oral squamous cell carcinoma. Geminin reactivity was widely distributed in high-grade dysplasia and oral squamous cell carcinoma rather than low-grade or no dysplastic cases. γ-H2A histone family member X was expressed in the superficial layer of oral epithelial precursor lesions and scattered carcinoma cells of oral squamous cell carcinoma. In oral squamous cell carcinoma, lower geminin expression was observed in recurrent cases. Geminin and γ-H2A histone family member X were associated with the degree of differentiation and mode of invasion. CONCLUSION: Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were correlated with oral carcinogenesis; these markers were associated with clinicopathological behaviors in oral squamous cell carcinoma.

Detection of human papillomavirus infection in oral squamous cell carcinoma: a cohort study of Japanese patients.

OBJECTIVE: Recent evidence suggests that human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is a separate HNSCC subgroup with distinct epidemiology, histopathological characteristics, therapeutic response to chemotherapy and radiation, and clinical outcome. This study aimed to investigate the role of HPV infection in oral squamous cell carcinoma (OSCC) and the correlation between HPV infection, tumor suppressor protein p16 expression, and clinicopathological features in Japanese patients. METHODS: In total, 174 OSCC specimens were examined for p16 levels by immunohistochemistry, and p16-positive OSCCs were analyzed for HPV DNA by in situ hybridization (ISH) and HPV genotypes by real-time PCR. The results were evaluated for the association with clinicopathological characteristics of OSCC patients. RESULTS: Twenty-four OSCC samples were found positive for p16 expression; all of them were well-differentiated tumors. P16 immunoreactivity was significantly associated with the invasion depth and tended to correlate with sex, site in the oral cavity, stromal reaction, TNM stage, and survival. HPV DNA was detected in 13 of 24 (54%) p16-positive OSCC by real-time PCR; HPV 16, 18, and other high-risk genotypes were the most prevalent. However, ISH failed to detect HPV DNA in p16-positive OSCCs. CONCLUSION: P16 immunoreactivity and HPV genotyping by real-time PCR may be useful markers of HPV infection in OSCC. However, although HPV-related OSCC showed good outcomes, HPV infection may have a minor role in oral oncogenesis in Japanese patients.

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

BACKGROUND: To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. METHODS: Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. RESULTS: Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. CONCLUSIONS: Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor.

The diagnostic utility of cytology specimen in a case of EWSR1::NFATC2 sarcoma.

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5' single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens.

Assessment of protein expression and gene status of human epidermal growth factor receptor (HER) family molecules in ameloblastomas.

BACKGROUND: To evaluate roles of human epidermal growth factor receptor (HER) family molecules in ameloblastomas, protein expression and gene status were analyzed in odontogenic tissues. METHODS: Sixty five ameloblastomas, 10 dental follicles, and 11 dentigerous cysts were immunohistochemically examined with antibodies against epidermal growth factor receptor (EGFR) and HER2, HER3, and HER4. Amplification of EGFR and HER2 was evaluated by chromogenic in situ hybridization (CISH). In 18 ameloblastomas, EGFR exons 19 and 21 were analyzed by direct DNA sequencing. RESULTS: Immunohistochemical reactivity for EGFR and HER2, HER3, and HER4 was detected in odontogenic epithelium. Expression of EGFR and HER4 was remarkable in these odontogenic tissues, as compared with that of HER2 and HER3. The level of HER2 immunoreactivity was significantly lower in ameloblastomas than in dental follicles and dentigerous cysts. Follicular ameloblastomas showed significantly higher expression of HER2 and HER4 than plexiform ameloblastomas. Reactivity for EGFR and HER3 was slightly stronger in recurrent ameloblastomas than in primary ameloblastomas. CISH did not reveal obvious amplification of EGFR or HER2 in ameloblastomas; however, EGFR and HER2 gene signals were significantly higher in follicular ameloblastomas than in plexiform ameloblastomas. Direct DNA sequencing of EGFR did not show any gene alteration in ameloblastomas. CONCLUSION: Expression of HER family molecules, especially EGFR and HER4, in odontogenic tissues suggests that growth signals mediated by these receptor molecules contribute to cell proliferation, survival, and differentiation in both normal and neoplastic odontogenic epithelial tissues. Some of these molecules might be useful for predicting outcomes in patients with ameloblastomas.

In vivo behavior of untreated and compressed concentrated growth factors as biomaterials in rabbits.

To characterize concentrated growth factors (CGFs) in vivo, we examined the degradation of implanted CGF in rabbits. Untreated CGF (U-CGF) and compressed CGF (C-CGF) were subcutaneously implanted into the dorsum. Histological analyses showed that the U-CGF and C-CGF induced very few inflammatory cells and that the U-CGF and C-CGF were subsequently degraded with dendritic invasion of granulation tissue. The C-CGF histopathologically remained for longer term than the U-CGF. Aggregated CD31+ and RAM11+ cells appeared in and around the implanted CGF. The number of macrophages and blood vessels in the CGF-implanted groups was greater than that in the sham group. There were more blood vessels in the U-CGF group than that in the C-CGF and sham group. We showed that CGF was degraded by macrophages in 4 weeks and enhanced angiogenesis with dendritically branching new capillaries. Therefore, the U-CGF and C-CGF can be clinically applied as a biomaterial inducing angiogenesis.

Expression of immunoregulatory molecules PD-L1 and PD-1 in oral cancer and precancerous lesions: A cohort study of Japanese patients.

OBJECTIVE: An association of the programmed cell death-1 (PD-1) and its ligand PD-L1 with various types of malignant tumors has been established. This study aimed to investigate the role of the PD-L1/PD-1 pathway in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPL). MATERIALS AND METHODS: We examined 106 OSCC and 79 OEPL specimens for PD-L1 and PD-1 expression by immunohistochemistry. The results were compared with clinicopathological features of OSCC patients. RESULTS: In OSCC and OEPL specimens, PD-L1 expression was detected predominantly in epithelial or carcinoma cells, whereas PD-1 expression was found mainly in infiltrating or stromal lymphocytes. Seventy-two OSCC (67.9%) and 21 OEPL (26.6%) specimens were positive for PD-L1, and 73 OSCC (68.9%) and 23 OEPL (29.2%) specimens were positive for PD-1. PD-L1 and PD-1 expression levels were significantly different between OEPL and OSCC specimens (P < 0.001). There were significant positive correlations between PD-L1 and PD-1 expression in OEPL and OSCC specimens (P < 0.001). PD-L1 and PD-1 immunoreactivity was significantly associated with tumor size (P < 0.05). PD-L1 and PD-1 immunoreactivity in cases with advanced TNM staging was significantly higher than that in low staging cases (P < 0.01). There were significant correlations between PD-L1 and PD-1 expression in OSCC specimens and pathological variables such as stromal lymphocytic reaction (P < 0.05) and invasion depth (P < 0.01). CONCLUSION: PD-L1 and PD-1 immunohistochemical status may be related to carcinogenesis, tumor progression, and prognosis in oral epithelial lesions. Agents targeting PD-1 and PD-L1 might be useful for OSCC treatment.

Peripheral clear cell variant of calcifying epithelial odontogenic tumor devoid of calcification.

A clear cell variant of calcifying epithelial odontogenic tumor (CCEOT) affecting an extraosseous site is described. A 60-year-old male patient presented with gingival swelling on the lingual side of the anterior mandible. The results of biopsy suggested clear cell odontogenic carcinoma, and marginal resection of the mandible was performed. The resected specimen was composed of eosinophilic and clear cells with deposits of amyloid-like material. The clear cells exhibited granules that were positive for PAS. There was no calcification in the resected lesion. Based on these features, the conclusive diagnosis was peripheral CCEOT without calcification. No signs of recurrence were evident after 3 years of follow-up.

A Case of Angiosarcoma Arising in Trunk of the Right Pulmonary Artery Clinically Simulating Pulmonary Thromboembolism.

Angiosarcoma arising in the pulmonary artery is extremely rare. We herein report a case of angiosarcoma arising in the pulmonary artery trunk of 71 year-old woman. This case was clinically diagnosed as pulmonary thromboembolism but angiosarcoma of the pulmonary artery should be considered as the differential diagnosis of unusual clinical manifestations of pulmonary thromboembolism because of the extremely poor prognosis of the lesion.