Detection of mesenchymal chondrosarcoma in pleural effusion with immunocytochemistry and determination of HEY1::NCOA2 fusion by next-generation sequencing on cell block.

Mesenchymal chondrosarcoma (MC) is a rare but extremely aggressive type of chondrosarcoma distinguished by the presence of both primitive mesenchymal cells and fully developed chondroid tissue. The identification of a biphasic morphology in pleural effusion, along with detection of the HEY1::NCOA2 fusion using next-generation sequencing, serve as vital indicators for an accurate diagnosis.

HEY1-NCOA2 expression modulates chondrogenic differentiation and induces mesenchymal chondrosarcoma in mice.

Mesenchymal chondrosarcoma affects adolescents and young adults, and most cases usually have the HEY1::NCOA2 fusion gene. However, the functional role of HEY1-NCOA2 in the development and progression of mesenchymal chondrosarcoma remains largely unknown. This study aimed to clarify the functional role of HEY1-NCOA2 in transformation of the cell of origin and induction of typical biphasic morphology of mesenchymal chondrosarcoma. We generated a mouse model for mesenchymal chondrosarcoma by introducing HEY1-NCOA2 into mouse embryonic superficial zone (eSZ) followed by subcutaneous transplantation into nude mice. HEY1-NCOA2 expression in eSZ cells successfully induced subcutaneous tumors in 68.9% of recipients, showing biphasic morphologies and expression of Sox9, a master regulator of chondrogenic differentiation. ChIP sequencing analyses indicated frequent interaction between HEY1-NCOA2 binding peaks and active enhancers. Runx2, which is important for differentiation and proliferation of the chondrocytic lineage, is invariably expressed in mouse mesenchymal chondrosarcoma, and interaction between HEY1-NCOA2 and Runx2 is observed using NCOA2 C-terminal domains. Although Runx2 knockout resulted in significant delay in tumor onset, it also induced aggressive growth of immature small round cells. Runx3, which is also expressed in mesenchymal chondrosarcoma and interacts with HEY1-NCOA2, replaced the DNA-binding property of Runx2 only in part. Treatment with the HDAC inhibitor panobinostat suppressed tumor growth both in vitro and in vivo, abrogating expression of genes downstream of HEY1-NCOA2 and Runx2. In conclusion, HEY1::NCOA2 expression modulates the transcriptional program in chondrogenic differentiation, affecting cartilage-specific transcription factor functions.

Genomic profiling identifies genes and pathways dysregulated by HEY1-NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis.

Mesenchymal chondrosarcoma is a rare, high-grade, primitive mesenchymal tumor. It accounts for around 2-10% of all chondrosarcomas and mainly affects adolescents and young adults. We previously described the HEY1-NCOA2 as a recurrent gene fusion in mesenchymal chondrosarcoma, an important breakthrough for characterizing this disease; however, little study had been done to characterize the fusion protein functionally, in large part due to a lack of suitable models for evaluating the impact of HEY1-NCOA2 expression in the appropriate cellular context. We used iPSC-derived mesenchymal stem cells (iPSC-MSCs), which can differentiate into chondrocytes, and generated stable transduced iPSC-MSCs with inducible expression of HEY1-NCOA2 fusion protein, wildtype HEY1 or wildtype NCOA2. We next comprehensively analyzed both the DNA binding properties and transcriptional impact of HEY1-NCOA2 expression by integrating genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and expression profiling (RNA-seq). We demonstrated that HEY1-NCOA2 fusion protein preferentially binds to promoter regions of canonical HEY1 targets, resulting in transactivation of HEY1 targets, and significantly enhances cell proliferation. Intriguingly, we identified that both PDGFB and PDGFRA were directly targeted and upregulated by HEY1-NCOA2; and the fusion protein, but not wildtype HEY1 or NCOA2, dramatically increased the level of phospho-AKT (Ser473). Our findings provide a rationale for exploring PDGF/PI3K/AKT inhibition in treating mesenchymal chondrosarcoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

NKX3.1 immunohistochemistry is highly specific for the diagnosis of mesenchymal chondrosarcomas: experience in the Australian population.

Mesenchymal chondrosarcoma (MC) is a rare sarcoma that typically arises in adolescents and young adults and characteristically harbours a HEY1-NCOA2 gene fusion. A recent study has shown that NKX3.1 immunohistochemistry (IHC) is highly specific and sensitive in MCs. NKX3.1 is a nuclear marker expressed in prostatic tissue and is widely used in most laboratories to determine prostatic origin of metastatic tumours. In the current study we investigated whether this stain can be used in the diagnostic workup of MC, as it may assist in triaging cases for further molecular testing, by assessing its expression in a cohort of MCs and in a wide spectrum of sarcoma types. Furthermore, we aimed to elucidate if expression of NKX3.1 by MCs is related to androgen receptor (AR) expression. We identified NKX3.1 positive nuclear staining in 9 of 12 individual patients of MC (n=20 of 25 samples when taking into account separate episodes). Four of the five negative specimens had been previously subjected to acid-based decalcification. NKX3.1 was negative in 536 samples from 16 non-MC sarcomas derived from largely tissue microarrays (TMAs). Overall, we identified 80% sensitivity and 100% specificity for NKX3.1 IHC in MCs. The sensitivity increased to 95.2% when acid-based decalcified specimens were excluded from the analysis. No correlation between NKX3.1 expression and AR IHC was identified. In summary, our findings indicate that NKX3.1 nuclear positivity is highly sensitive and specific for MC, provided that ethylenediaminetetraacetic acid (EDTA)-based rather than acid-based decalcification is used for sample processing. NKX3.1 IHC in the right clinical and histopathological setting can potentially be sufficient for the diagnosis of MC, reserving molecular confirmation only for equivocal cases.

NKX3.1 immunoreactivity is not identified in mesenchymal chondrosarcoma: a 25-case cohort study.

AIMS: Mesenchymal chondrosarcoma (MC) is characterised typically by a bimorphic microscopic appearance of islands of a well-differentiated cartilaginous component, admixed with a primitive small cell component, which commonly expresses CD99 and NKX2.2. Given the variable relative abundance of each component and histological overlap with other small round cell tumours, the diagnosis can be challenging, especially in a limited sample. A distinctive gene fusion between HEY1 (located in 8q21) and NCOA2 (located in 8q13) was identified in MC, but the downstream molecular events are unknown. NKX3.1 (coding gene located in 8p21.1) was recently reported to be expressed in a small number of MC cases. The purpose of this study was to evaluate the potential diagnostic utility of NKX3.1 immunohistochemistry in MC. METHODS AND RESULTS: We evaluated sections from 25 cases of MC, including 20 extraskeletal and five osseous. The tumour affected nine females and 16 males, with a median age of 34 years (age range = 7-82 years). Two different rabbit antibodies against NKX3.1 (monoclonal and polyclonal) were used for immunohistochemistry. However, no immunoreactivity was observed with either of the antibodies in all 25 (100%) MC. CONCLUSIONS: NKX3.1 immunoreactivity was not identified in our cohort. Clonality of the antibody could not explain the negativity.

NKX3.1 a useful marker for mesenchymal chondrosarcoma: An immunohistochemical study.

INTRODUCTION: Mesenchymal chondrosarcoma is a rare subtype of chondrosarcoma. The tumor has a characteristic bimorphic pattern with areas of poorly differentiated small round cell component and interspersed islands of well differentiated hyaline cartilage. Histological diagnosis of mesenchymal chondrosarcoma is very challenging especially in small biopsies when tumor presents with little cartilaginous component. In such cases, it is very difficult to distinguish mesenchymal chondrosarcoma from other round blue cell tumors like Ewing's sarcoma, rhabdomyosarcoma, small cell osteosarcoma and desmoplastic round blue cell tumor. Immunohistochemically, mesenchymal chondrosarcoma stains positive for NKX2.2, CD99, S100 and SOX9. This immunoprofile is non-specific and overlaps with other round blue cell tumors. Till recently, there was no reliable immunohistochemical marker to differentiate mesenchymal chondrosarcoma from other round blue cell tumors. NKX3.1, though widely used as a diagnostic biomarker for prostatic adenocarcinoma, has been recently proposed by Yoshida et al. (2020) as a unique marker of mesenchymal chondrosarcoma and EWSR1-NFATC2 sarcoma. OBJECTIVE: The aim of our study was to further explore utility of NKX3.1 as a diagnostic marker of mesenchymal chondrosarcoma. MATERIAL & METHODS: We applied NKX3.1 immunohistochemistry to 21 cases of mesenchymal chondrosarcoma and 32 cases of other round blue cell tumors. RESULTS: 14 out of 21 cases (66.7%) of mesenchymal chondrosarcoma stained positive for NKX3.1 with nuclear expression in small round component. Cartilaginous component was predominantly negative. All other round blue cell tumors showed negative results. CONCLUSION: Based on our study results we suggest that NKX3.1 is a useful immunohistochemical marker in differentiating mesenchymal chondrosarcoma from its histological mimics.

Mesenchymal chondrosarcomas showing immunohistochemical evidence of rhabdomyoblastic differentiation: a potential diagnostic pitfall.

The diagnosis of mesenchymal chondrosarcoma, a distinctive biphasic malignant neoplasm harboring the HEY1-NCOA2 gene fusion and consisting of primitive round to spindled cells admixed with foci of relatively mature hyaline cartilage, is usually straightforward by morphologic evaluation alone. However, in the setting of a limited biopsy, specimens lacking cartilage generate a broad differential diagnosis, encompassing a variety of other primitive sarcomas, including spindle cell/sclerosing rhabdomyosarcoma. Although a small number of cases of mesenchymal chondrosarcoma with aberrant skeletal muscle marker expression have been reported, pathologists are largely unaware of this potential diagnostic pitfall. We report 6 additional cases of mesenchymal chondrosarcoma showing expression of multiple skeletal muscle markers, including one case initially misdiagnosed as "spindle cell/sclerosing rhabdomyosarcoma" on needle biopsy. Awareness of this phenomenon and judicious application of molecular diagnostic testing for the HEY1-NCOA2 fusion are critical to avoid misclassification of mesenchymal chondrosarcoma as rhabdomyosarcoma, with potentially adverse patient impact.

Intracranial Extraskeletal Mesenchymal Chondrosarcoma.

BACKGROUND: Intracranial extraskeletal mesenchymal chondrosarcomas (MCSs) are rare tumors accounting for <0.16% of intracranial tumors. They are usually described as occurring in the age group of 20-30 years and are commonly found in the frontoparietal region arising from the falx and surrounding dura. We describe 3 cases at varying ages, locations, and outcomes. CASE DESCRIPTION: A 42-year-old woman with anterior one third falx-based lesion; a 7-year-old boy with mid one third falcine lesion with hyperostosis of bone, presenting in comatose stage; and a 52-year-old woman with left posterior lateral frontal dural-based lesion are presented. Histopathology of all cases was confirmed with immunohistochemistry. All patients underwent complete excision but had varying outcomes. CONCLUSIONS: Intracranial MCSs are rare malignant tumors with poor prognosis. Because of their rarity, it may not be considered as a differential diagnosis and it is often misdiagnosed radiologically, but the importance lies in the need of radical excision. The potential effect of postoperative radiotherapy and chemotherapy is controversial. This article sheds some light on variable prognosis of this tumor.

Are meningeal hemangiopericytoma and mesenchymal chondrosarcoma the same?: a study of HEY1-NCOA2 fusion.

OBJECTIVES: Meningeal hemangiopericytoma (HPC) and mesenchymal chondrosarcoma are aggressive neoplasms with a propensity to involve the meninges and dura. In addition to similar clinical presentations, both meningeal HPC and mesenchymal chondrosarcoma share overlapping morphologic features, including ovoid cells, variable collagen deposition, and a branching vascular pattern. Recently, a novel HEY1-NCOA2 fusion was reported as a recurrent event in mesenchymal chondrosarcomas. METHODS: Thirteen mesenchymal chondrosarcomas and 18 meningeal HPCs were identified from surgical pathology archives, and the tumors were evaluated for HEY1-NCOA2 fusion with reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: HEY1-NCOA2 fusion transcript was detected in all six cases of mesenchymal chondrosarcoma but in none of the meningeal HPC cases (0/11) that were evaluable with RT-PCR. CONCLUSIONS: These results show that (1) meningeal HPC and mesenchymal chondrosarcoma are distinct at the molecular level, and (2) the identification of HEY1-NCOA2 can be used as an auxiliary diagnostic tool to differentiate these entities.

Screening for potential targets for therapy in mesenchymal, clear cell, and dedifferentiated chondrosarcoma reveals Bcl-2 family members and TGFß as potential targets.

The mesenchymal, clear cell, and dedifferentiated chondrosarcoma subtypes are extremely rare, together constituting 10% to 15% of all chondrosarcomas. Their poor prognosis and lack of efficacious treatment emphasizes the need to elucidate the pathways playing a pivotal role in these tumors. We constructed tissue microarrays containing 42 dedifferentiated, 23 clear cell, and 23 mesenchymal chondrosarcomas and performed immunohistochemistry to study the expression of growth plate-signaling molecules and molecules shown to be involved in conventional chondrosarcoma. We observed high expression of SOX-9 and FGFR-3, as well as aberrant cellular localization of heparan sulfate proteoglycans, in all subtypes. TGFß signaling through p-SMAD2 and PAI-1 was highly active in all chondrosarcoma subtypes, which suggests that TGFß inhibitors as a possible therapeutic strategy in rare chondrosarcoma subtypes. As in conventional chondrosarcoma, antiapoptotic proteins (Bcl-2, and/or Bcl-xl) were highly expressed in all subtypes. Inhibition with the BH-3 mimetic ABT-737 rendered dedifferentiated chondrosarcoma cell lines sensitive to doxorubicin or cisplatin. Our data indicate that antiapoptotic proteins may play an important role in chemoresistance, suggesting a promising role for targeting Bcl-2 family members in chondrosarcoma treatment, irrespective of the subtype.

Giant primary mesenchymal chondrosarcoma of the lung: case report and review of literature.

Mesenchymal chondrosarcoma, a rare malignant tumor, was predominantly occurring in the bone and may involve somatic soft tissue but it is extremely rare in the lung.We report the case of a 20-year-old female who presented with a 2-month history of irritant nonproductive cough and chest pain. The histopathologic examination revealed the tumor composed of atypical undifferentiated small cells and islands of matured chondroid matrix typically presented as bimorphic appearances. Immunohistochemical examination revealed that the tumor cells were positive for vimentin and CD99 for all components, and to S-100 limited to the areas of cartilage. In addition, previously reported cases of primary lung mesenchymal chondrosarcoma were reviewed, and the relevant clinical knowledge regarding its clinical manifestations, diagnosis, and treatment were discussed.

Diagnostic pitfall in the diagnosis of mesenchymal chondrosarcoma arising in the central nervous system.

Mesenchymal chondrosarcoma is a rare aggressive neoplasm typically affecting the bones of young adults. It may also arise in somatic soft tissue, the CNS and other organs. It has a characteristic biphasic histological pattern composed of highly undifferentiated small round cells and islands of well-differentiated hyaline cartilage. We report a case of mesenchymal chondrosarcoma arising from the right tentorium cerebelli in a 21-year-old woman with symptoms relating to mass effect. Histological examination demonstrated a purely small round cell appearance in a specimen obtained during partial resection at an outside institution, leading to an erroneous diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (PNET). The diagnosis of mesenchymal chondrosarcoma was made only after tissue obtained during a definitive complete macroscopic removal involving the regional tentorium cerebelli, transverse and sigmoid dural venous sinuses which showed a prominent cartilaginous component. We discuss the features of mesenchymal chondrosarcoma arising in the CNS, the important differential diagnoses of small round-cell tumors within the CNS, and the differentiating features of mesenchymal chondrosarcoma from Ewing sarcoma/PNET, medulloblastoma, hemangiopericytoma, monophasic synovial sarcoma and atypical teratoid/rhabdoid tumour.

Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data.

Cancer gene fusions that encode a chimeric protein are often characterized by an intragenic discontinuity in the RNA\expression levels of the exons that are 5' or 3' to the fusion point in one or both of the fusion partners due to differences in the levels of activation of their respective promoters. Based on this, we developed an unbiased, genome-wide bioinformatic screen for gene fusions using Affymetrix Exon array expression data. Using a training set of 46 samples with different known gene fusions, we developed a data analysis pipeline, the "Fusion Score (FS) model", to score and rank genes for intragenic changes in expression. In a separate discovery set of 41 tumor samples with possible unknown gene fusions, the FS model generated a list of 552 candidate genes. The transcription factor gene NCOA2 was one of the candidates identified in a mesenchymal chondrosarcoma. A novel HEY1-NCOA2 fusion was identified by 5' RACE, representing an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. RT-PCR or FISH evidence of this HEY1-NCOA2 fusion was present in all additional mesenchymal chondrosarcomas tested with a definitive histologic diagnosis and adequate material for analysis (n = 9) but was absent in 15 samples of other subtypes of chondrosarcomas. We also identified a NUP107-LGR5 fusion in a dedifferentiated liposarcoma but analysis of 17 additional samples did not confirm it as a recurrent event in this sarcoma type. The novel HEY1-NCOA2 fusion appears to be the defining and diagnostic gene fusion in mesenchymal chondrosarcomas.

[Clinicopathologic and immunohistochemical study of 23 cases of mesenchymal chondrosarcoma].

OBJECTIVE: To study the clinicopathologic and immunohistochemical features of mesenchymal chondrosarcoma. METHODS: The clinical and histologic features of 23 cases of mesenchymal chondrosarcoma were analyzed. Immunohistochemical study was also performed in 14 of the cases. RESULTS: The age of patients ranged from 12 to 47 years. Fourteen of them occurred in males. Thirteen cases involved the bony skeleton and 5 cases affected the soft tissue. The patients presented with pain and/or swelling. Histologically, the tumor consisted of a mixture of undifferentiated small round cells and hyaline cartilage. Transition between the two components was demonstrated and growth plate-like cartilage was observed. Immunohistochemical study showed that the small round cells were positive for Sox9 (14/14), CD99 (12/14), vimentin (6/14), CD56 (4/14), CD57 (4/14), neuron-specific enolase (3/14) and desmin(1/14). They were negative for Coll-II, S-100 protein, epithelial membrane antigen, pan-cytokeratin, synaptophysin, chromogranin A, CD34 and c-erbB2. CONCLUSIONS: Mesenchymal chondrosarcoma is a rare malignant tumor. Thorough histologic examination, when coupled with immunohistochemical findings, is helpful in arriving at a correct diagnosis.

Reappraisal of mesenchymal chondrosarcoma: novel morphologic observations of the hyaline cartilage and endochondral ossification and beta-catenin, Sox9, and osteocalcin immunostaining of 22 cases.

Mesenchymal chondrosarcoma, a rare malignant round cell and hyaline cartilage tumor, is most commonly intraosseous but can occur in extraskeletal sites. We intensively observed the morphology and applied Sox9 (master regulator of chondrogenesis), beta-catenin (involved in bone formation, thought to inhibit chondrogenesis in a Sox9-dependent manner), and osteocalcin (a marker for osteoblastic phenotype) to 22 central nervous system and musculoskeletal mesenchymal chondrosarcoma. Cases of mesenchymal chondrosarcoma were retrieved and reviewed from our files. Immunohistochemistry and follow-up were obtained on mesenchymal chondrosarcoma and tumor controls. Twenty-two mesenchymal chondrosarcomas included 5 central nervous system (all female; mean age, 30.2; mean size, 7.8 cm; in frontal lobe [n = 4] and spinal cord [n = 1]) and 17 musculoskeletal (female-male ratio, 11:6; mean age, 31.1; mean size, 6.2 cm; 3 each of humerus and vertebrae; 2 each of pelvis, rib, tibia, neck soft tissue; one each of femur, unspecified bone, and elbow soft tissue). The hyaline cartilage in most tumors revealed a consistent linear progression of chondrocyte morphology, from resting to proliferating to hypertrophic chondrocytes. Sixty-seven percent of cases demonstrated cell death and acquired osteoblastic phenotype, cells positive for osteocalcin at the site of endochondral ossification. Small round cells of mesenchymal chondrosarcoma were negative for osteocalcin. SOX9 was positive in both components of 21 of 22 cases of mesenchymal chondrosarcoma. beta-Catenin highlighted rare nuclei at the interface between round cells and hyaline cartilage in 35% cases. Control skull and central nervous system cases were compared, including chondrosarcomas and small cell osteosarcoma, the latter positive for osteocalcin in small cells. Mesenchymal chondrosarcoma demonstrates centrally located hyaline cartilage with a linear progression of chondrocytes from resting to proliferative to hypertrophic, which undergoes endochondral ossification, recapitulating growth plate cartilage and suggesting that this component of mesenchymal chondrosarcoma may be a differentiated (benign or metaplastic) component of a malignant metastasizing tumor. This hyaline cartilage component is morphologically different from cartilage of control chondrosarcoma. Mesenchymal chondrosarcoma can be separated from small cell osteosarcoma, using Sox 9 for cartilage and osteocalcin for osteoblastic phenotype. Rare nuclear beta-catenin expression at the interface between hyaline cartilage and small round cells potentially implicates the APC/Wnt pathway during endochondral ossification in morphologically benign hyaline cartilage component of mesenchymal chondrosarcoma.

Immunoprofile of mesenchymal chondrosarcoma: aberrant desmin and EMA expression, retention of INI1, and negative estrogen receptor in 22 female-predominant central nervous system and musculoskeletal cases.

Mesenchymal chondrosarcoma is a rare malignant tumor in the differential diagnosis of other small, round blue cell tumors, including atypical teratoid tumor in the central nervous system (CNS) and rhabdomyosarcoma in the musculoskeletal (MSK) locations. We reviewed the morphology of CNS and MSK cases and applied a panel of immunostains. Archival cases were pulled from our files. Immunohistochemistry and follow-up were obtained. Twenty-two cases included 5 CNS (all female; mean age, 30.2) and 17 MSK (11 female and 6 male; mean age, 31.1). Both CNS and MSK examples had similar round cells, staghorn vascular pattern, increased mitotic activity, and centrally located hyaline cartilage islands. The CNS examples demonstrated more spindling and the MSK cases more necrosis. INI1 was retained in all tumors studied. Epithelial membrane antigen (EMA) and desmin were expressed focally in 35% and 50% of cases, respectively. The round cells of all cases were negative for MyoD1, myogenin, smooth muscle actin (SMA), glial fibrillary acid protein (GFAP), keratins, and estrogen receptor, as well as a panel of other antiobodies. Eighty percent of patients with follow-up had pulmonary metastases and/or died within a mean of 5 years. The CNS and MSK mesenchymal chondrosarcoma predominantly affects adult females with poor prognosis. There are only subtle morphologic differences between the CNS and MSK groups. By immunohistochemistry, mesenchymal chondrosarcoma occasionally expresses aberrant desmin and EMA but is negative for SMA, myogenin MyoD1, GFAP, and keratins, refuting true smooth or skeletal muscle, epithelial, or meningothelial phenotype. Retained INI1 separates these tumors from atypical teratoid tumor. Despite marked female predominance in our series, estrogen receptor is negative in mesenchymal chondrosarcoma.