Diagnostic value of MDM2 RNA in situ hybridization for low-grade osteosarcoma: Consistency comparison of RNA in situ hybridization, fluorescence in situ hybridization, and immunohistochemistry.

Detection of MDM2 gene amplification via fluorescence in situ hybridization (FISH) and MDM2 overexpression by immunohistochemistry (IHC) have been utilized for the diagnosis of low-grade osteosarcoma (LGOS). The aim of this study was to evaluate the diagnostic value of MDM2 RNA in situ hybridization (RNA-ISH) and compare this assay with MDM2 FISH and IHC in distinguishing LGOS from its histologic mimics. MDM2 RNA-ISH, FISH and IHC were performed on nondecalcified samples of 23 LGOSs and 52 control cases. Twenty (20/21, 95.2%) LGOSs were MDM2-amplified, and two cases failed in FISH. All control cases were MDM2-nonamplified. All 20 MDM2-amplified LGOSs and one MDM2-nonamplified LGOS harboring TP53 mutation and RB1 deletion showed positivity for RNA-ISH. Fifty of the 52 (96.2%) control cases were negative for RNA-ISH. The diagnostic sensitivity and specificity of MDM2 RNA-ISH were 100.0% and 96.2%, respectively. Nineteen of the 23 LGOSs were evaluated by MDM2 RNA-ISH and FISH in decalcified samples simultaneously. All decalcified LGOSs failed in FISH and most samples (18/19) were no staining in RNA-ISH. Fifteen (15/20, 75%) MDM2-amplified LGOSs were positive for IHC and 96.2% (50/52) of control cases were negative. The sensitivity of RNA-ISH (100%) was higher than that of IHC (75%). In conclusion, MDM2 RNA-ISH has great value for the diagnosis of LGOS, with excellent consistency with FISH and better sensitivity than IHC. Acid decalcification still has an adverse impact on RNA. Some MDM2-nonamplified tumors may show positivity for MDM2 RNA-ISH, which needs to be analyzed comprehensively in combination with clinicopathological features.

Case report: Primary pleural low-grade fibromyxoid sarcoma in a 4-year-old boy with molecular confirmation.

Low-grade fibromyxoid sarcoma (LGFMS) is a rare malignant fibroblastic tumor, principally affecting the deep tissues of the proximal trunk and extremities in young adults. However, primary pleural LGFMS is extremely rare, and only three cases have been reported in the previous English literature without genetic confirmation. Furthermore, the historical pleural LGFMS cases were all adults, and the primary pleural LGFMS in children has never been reported to date. Here, we presented a primary pleural LGFMS in a 4-year-old boy with detailed clinical, pathological, and molecular results. Histologically, the current tumor showed typical alternating collagenous and myxoid areas, containing spindled or oval tumor cells arranged in a whorled and short fascicular pattern. In some areas, the tumor cells exhibited moderate atypia, and mitotic figures were identified but without the identification of giant collagen rosettes. Immunohistochemically, all the neoplastic cells showed strong and diffuse positivity for MUC4. Genetically, FUS gene rearrangement was revealed by fluorescence in-situ hybridization (FISH), and subsequently, next-generation sequencing (NGS) and polymerase chain reaction (PCR) further demonstrated the FUS::CREB3L2 fusion transcript. To the best of our knowledge, this is the first case of primary pleural LGFMS with the identification of FUS gene rearrangement and FUS::CREB3L2 fusion in a 4-year-old child. Our study expands the age range of pleural LGFMS and highlights the combination of morphological, immunohistochemical, and molecular analyses in such challenging cases.

Primary intrathoracic liposarcomas: A clinicopathologic and molecular study of 43 cases in one of the largest medical centers of China.

Introduction: Primary intrathoracic liposarcoma is extremely rare, and most published series lack genetic analyses. The aim of our study is to better understand the clinicopathologic and genetic features of these rare lesions. Materials and methods: Forty-three primary intrathoracic liposarcomas were identified and most cases were analyzed by systematic genetic studies, including fluorescence in situ hybridization (FISH), whole-exome sequencing (WES), and Sanger sequencing. Results: This series included 27 males and 16 females (ratios, 1.68:1) aged 24-73 years (median, 53 years). Tumors mainly occurred in the mediastinum (n=23, 53.5%), followed by pleural cavity (n=16, 37.2%) and lung (n=4, 9.3%). The study included 21 well-differentiated liposarcomas (WDLs), 19 dedifferentiated liposarcomas (DDLs), 2 myxoid pleomorphic liposarcomas (MPLs) and 1 pleomorphic liposarcoma (PL), without identification of myxoid liposarcoma. FISH analysis identified MDM2 amplification in 17 of 18 WDLs (94.4%) and all DDLs (16/16, 100.0%). The MDM2-nonamplified WDL was CDK4-nonamplified but FRS2-amplified. WES and Sanger sequencing found somatic TP53 mutation in the 2 MPLs. Follow-up information was available for 33 of 38 cases (86.8%). Thirteen patients (39.4%) showed no evidence of disease, 10 patients (30.3%) were alive with disease, and 8 patients (24.2%) died of disease. Fourteen cases developed recurrence and 1 with metastasis. Conclusions: WDL/DDL was the overwhelming subtype in this location, followed by MPL and PL. Analysis of the FRS2 gene, in combination with MDM2 and other genes of 12q13-15, may more precisely characterize WDL/DDLs. MPL is the most fatal subtype of this site. Further studies are needed to explore the role of TP53 in the pathogenesis of MPL.

Unusual split green-orange signals in USP6 fluorescence in situ hybridization in a malignant peripheral nerve sheath tumor with a novel NF1-SCIMP fusion: a potential diagnostic pitfall.

Deletion of the neurofibromatosis 1 (NF1) gene is common, but NF1 rearrangement or fusion has rarely been reported in peripheral nerve sheath tumors. Here, we present a case of malignant peripheral nerve sheath tumor (MPNST) in a 36-year-old Chinese female. Histologically, the lesion was composed of spindle cells with moderate atypia, immature bone, and atypical cartilage elements. Fluorescence in situ hybridization (FISH) for USP6 revealed green-orange split signals, strongly suggesting the presence of USP6 rearrangement. Subsequent next-generation sequencing-based technology analyses revealed t(17,17) (p13.2, q11.2) intrachromosomal translocation resulting in a novel NF1-SCIMP fusion gene along with NF1 deletion. However, USP6 fusion was not identified. To the best of our knowledge, this is the first case with a confirmed NF1 gene fusion partner in a peripheral nerve sheath tumor. Notably, rearrangement of the SCIMP may cause a pitfall in the interpretation of USP6 FISH results.

Expression of FRS2 in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: an immunohistochemical analysis of 182 cases with genetic data.

BACKGROUND: The fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4 within the 12q13-15 chromosomal region. FRS2 gene was recently found to be consistently amplified in atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL), suggesting the detection of FRS2 amplification could be a diagnostic tool for ALT/WDL/DDLs. However, the expression of FRS2 protein and diagnostic value of FRS2 immunohistochemistry (IHC) has not been evaluated in a large cohort of ALT/WDL/DDLs. METHODS: A SNOMED search of hospital surgical pathology files from January 2007 to July 2020 identified 182 ALT/WDL/DDLs with available materials. FRS2 fluorescence in situ hybridization (FISH) and IHC were performed on 182 ALT/WDL/DDLs and 64 control samples. The expression of FRS2 was also compared with that of classic immunomarkers (MDM2 and CDK4) of this tumor entity. RESULTS: This study included 91 ALT/WDLs and 91 DDLs. The FISH results showed 172 of 182 (94.5%) cases were FRS2-amplified, and 10 cases were FRS2-nonamplified. Immunostaining results showed 171 (94.0%) ALT/WDL/DDLs were positive for FRS2 and 11 cases (6.0%) were FRS2-immunonegative. In 172 FRS2-amplified cases, 166 (96.5%) were FRS2-immunopositive, and 6 (3.5%) were negative. Among 10 FRS2-nonamplified ALT/WDL/DDL cases, 5 cases were FRS2-immunonegative, and 5 tumors displayed 1+ staining for this marker. In 64 control cases, none of them exhibited FRS2 amplification. Forty-seven (73.5%) control cases were negative for FRS2 immunostaining, while 17 cases (26.5%) were FRS2-immunopositive. Fifteen of these false positive samples (15/17, 88.2%) showed 1+ positivity and only 2 cases (2/17, 11.8%) displayed 2+ positivity. In ALT/WDL/DDLs, the sensitivity of FRS2 immunostaining was slightly lower than MDM2 (FRS2 vs. MDM2: 94.0% vs 100.0%) and CDK4 (FRS2 vs. CDK4: 94.0% vs 97.0%). However, the specificity of FRS2 (73.5%) was slightly higher than that of MDM2 (67.8%) and CDK4 (64.4%). CONCLUSION: This study indicated that FRS2 IHC had relatively good consistency with FRS2 FISH, suggesting that FRS2 immunostaining could be utilized as an additional screening tool for the diagnosis of ALT/WDL/DDL. It must be emphasized that MDM2/CDK4/FRS2 especially MDM2 FISH remains the gold standard and the most recommended method to diagnose this entity.

Consistent Amplification of FRS2 and MDM2 in Low-grade Osteosarcoma: A Genetic Study of 22 Cases With Clinicopathologic Analysis.

Low-grade osteosarcoma (LGOS) encompasses low-grade central osteosarcoma (LGCOS) and parosteal osteosarcoma (POS). LGOSs are characterized by a supernumerary ring and giant rod chromosomes containing the 12q13-15 amplicon. The fibroblast growth factor receptor substrate 2 (FRS2) gene is located close to MDM2 and CDK4. Recent studies identified consistent amplification of FRS2 gene in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma. The aim of this study was to evaluate the frequency of FRS2 amplification and its relationship with the clinicopathologic features of LGOSs. The amplification of FRS2 and MDM2 genes were analyzed by fluorescence in situ hybridization using 22 LGOSs (3 LGCOSs, 14 classic POSs, and 5 dedifferentiated POSs) and 85 control samples of bone and soft tissue. The clinicopathologic features of the 22 LGOSs were described. Amplification of FRS2 was detected in 21/22 (95%) of the LGOSs, including 3 (100%) LGCOSs and 18 (95%) POSs. All 22 LGOSs showed MDM2 amplification (100%). The only MDM2/FRS2 LGOS was dedifferentiated POS (the dedifferentiated component was conventional osteosarcoma). In the control group, all of the atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcomas (DDLs) (10/10, 100%) were FRS2-amplified, whereas the remaining 75 control cases were FRS2-nonamplified. These findings indicate that the FRS2 gene is consistently amplified in classic and dedifferentiated LGOSs but not in their histologic mimics. These results offer another avenue for investigating the biology of LGOSs. Whether FRS2-nonamplified tumors exhibit unusual clinicopathologic features needs further investigation. Some so-called "high-grade osteosarcomas harboring 12q13-15 amplification" may be unrecognized dedifferentiated LGOSs.

Amplification of FRS2 in atypical lipomatous tumour/well-differentiated liposarcoma and de-differentiated liposarcoma: a clinicopathological and genetic study of 146 cases.

AIMS: The aim of this study was to evaluate the frequency of FRS2 amplification and its relationship with the clinicopathological features of atypical lipomatous tumour (ALT)/well-differentiated liposarcoma (WDL)/de-differentiated liposarcoma (DDL). METHODS AND RESULTS: FRS2 and MDM2 fluorescence in-situ hybridisation (FISH) was performed on 146 tumours (70 ALT/WDLs and 76 DDLs). One hundred and eight control samples were included for FRS2 analysis. FRS2 amplification was detected in 136 of 146 (93.2%) ALT/WDL/DDLs, including 63 ALT/WDLs and 73 DDLs. A higher FRS2/CEP12 ratio was observed in DDLs than in ALT/WDLs (P = 0.0005). The FRS2/CEP12 ratio of peripheral tumours was lower than that of central tumours (P = 0.00004). All the ALT/WDL/DDLs showed MDM2 amplification (100%). The MDM2+ /FRS2- series included seven ALT/WDLs and three DDLs. Four of seven (57.1%) MDM2+ /FRS2- ALT/WDLs occurred in peripheral sites, slightly higher than the percentage of MDM2+ /FRS2+ ALT/WDLs (28 of 63, 44.4%). All the three MDM2+ /FRS2- DDLs (100%) were peripheral tumours, a much higher proportion than that of MDM2+ /FRS2+ DDLs (10 of 73, 13.7%). A high percentage of homologous pleomorphic liposarcoma-like DDLs (two of three) were observed in the MDM2+ /FRS2- group. In the control group all the parosteal osteosarcomas (five of five, 100%) were FRS2 amplified, whereas the remaining 103 samples were FRS2 non-amplified. CONCLUSIONS: These findings suggest that FRS2 is amplified consistently in ALT/WDL/DDLs and offer another avenue for the investigation of the biology of this tumour group. MDM2+ /FRS2- cases seem to be associated with certain clinicopathological features, and further investigation is needed.

Synovial sarcoma showing loss of a green signal in SS18 fluorescence in situ hybridization: a clinicopathological and molecular study of 12 cases.

The phenomenon of losing a green signal in synovial sarcoma (SS) using the SS18 break-apart probe by fluorescence in situ hybridization (FISH) has been poorly described. In this study, 12 SS with missing a green signal were identified. This series included 7 males and 5 females, aged 17 to 69 years (median, 38.5 years). The tumors involved the extremities (50%), mediastinum (16.7%), hypopharynx (8.3%), neck (8.3%), thyroid (8.3%), and retroperitoneum (8.3%). The tumors were classified as monophasic SS (58.3%) and poorly differentiated SS (41.7%). An anaplastic SS showing features of pleomorphic sarcoma was observed. Immunostaining for TLE1, BCL2, CD99, epithelial membrane antigen, cytokeratin (AE1/AE3), cytokeratin 7, S-100 protein, and CD34 was consistent with typical SS. In FISH, all the tumors showed the pattern of 1 to 3 fused signal(s) with 1 to 3 red signal(s), without corresponding a green signal. The fusion transcripts included SS18-SSX1 (8/10, 80%) and SS18-SSX2 (2/10, 20%) fusions. Median and 5-year overall survival were 19.1 months and 43.6%, respectively. In conclusion, we reported a series of SS losing a green signal in the SS18 FISH assay. We propose that this variant FISH pattern should be interpreted as a peculiar unbalanced rearrangement of the SS18 gene and subsequent SS18-SSX fusion test should be recommended. The cases in this study seem to show some unusual clinicopathological features, including unusual locations, higher proportions of poorly differentiated SS, and aggressive clinical course. However, whether this variant FISH pattern is associated with peculiar clinicopathologic features awaits larger series.