Machine Learning-Supported Diagnosis of Small Blue Round Cell Sarcomas Using Targeted RNA Sequencing.

Small blue round cell sarcomas (SBRCSs) are a heterogeneous group of tumors with overlapping morphologic features but markedly varying prognosis. They are characterized by distinct chromosomal alterations, particularly rearrangements leading to gene fusions, whose detection currently represents the most reliable diagnostic marker. Ewing sarcomas are the most common SBRCSs, defined by gene fusions involving EWSR1 and transcription factors of the ETS family, and the most frequent non-EWSR1-rearranged SBRCSs harbor a CIC rearrangement. Unfortunately, currently the identification of CIC::DUX4 translocation events, the most common CIC rearrangement, is challenging. Here, we present a machine-learning approach to support SBRCS diagnosis that relies on gene expression profiles measured via targeted sequencing. The analyses on a curated cohort of 69 soft-tissue tumors showed markedly distinct expression patterns for SBRCS subgroups. A random forest classifier trained on Ewing sarcoma and CIC-rearranged cases predicted probabilities of being CIC-rearranged >0.9 for CIC-rearranged-like sarcomas and <0.6 for other SBRCSs. Testing on a retrospective cohort of 1335 routine diagnostic cases identified 15 candidate CIC-rearranged tumors with a probability >0.75, all of which were supported by expert histopathologic reassessment. Furthermore, the multigene random forest classifier appeared advantageous over using high ETV4 expression alone, previously proposed as a surrogate to identify CIC rearrangement. Taken together, the expression-based classifier can offer valuable support for SBRCS pathologic diagnosis.

Small cell osteosarcoma versus fusion-driven round cell sarcomas of bone: retrospective clinical, radiological, pathological, and (epi)genetic comparison with clinical implications.

Small cell osteosarcoma (SCOS), a variant of conventional high-grade osteosarcoma (COS), may mimic fusion-driven round cell sarcomas (FDRCS) by overlapping clinico-radiological and histomorphological/immunohistochemical characteristics, hampering accurate diagnosis and consequently proper therapy. We retrospectively analyzed decalcified formalin-fixed paraffin-embedded (FFPE) samples of 18 bone tumors primarily diagnosed as SCOS by methylation profiling, fusion gene analysis, and immunohistochemistry.In eight cases, the diagnosis of SCOS was maintained, and in 10 cases it was changed into FDRCS, including three Ewing sarcomas (EWSR1::FLI1 in two cases and no identified fusion gene in the third case), two sarcomas with BCOR alterations (KMT2D::BCOR, CCNB3::BCOR, respectively), three mesenchymal chondrosarcomas (HEY1::NCOA2 in two cases and one case with insufficient RNA quality), and two sclerosing epithelioid fibrosarcomas (FUS::CREBL3 and EWSR1 rearrangement, respectively).Histologically, SCOS usually possessed more pleomorphic cells in contrast to the FDRCS showing mainly monomorphic cellular features. However, osteoid was seen in the latter tumors as well, often associated with slight pleomorphism. Also, the immunohistochemical profile (CD99, SATB2, and BCOR) overlapped.Clinically and radiologically, similarities between SCOS and FDRCS were observed, with by imaging only minimal presence or lack of (mineralized) osteoid in most of the SCOSs.In conclusion, discrimination of SCOS, epigenetically related to COS, versus FDRCS of bone can be challenging but is important due to different biology and therefore therapeutic strategies. Methylation profiling is a reliable and robust diagnostic test especially on decalcified FFPE material. Subsequent fusion gene analysis and/or use of specific immunohistochemical surrogate markers can be used to substantiate the diagnosis.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

CIC-Rearranged Sarcoma.

CIC-rearranged sarcoma is a rare type of small round cell sarcoma. The tumors often affect the deep soft tissues of patients in a wide age range. They are highly aggressive, respond poorly to chemotherapy, and have a worse outcome than Ewing sarcoma. CIC-rearranged sarcoma has characteristic and recognizable histology, including lobulated growth, focal myxoid changes, round to epithelioid cells, and minimal variation of nuclear size and shape. Nuclear ETV4 and WT1 expression are useful immunohistochemical findings. CIC fusion can be demonstrated using various methods; however, even next-generation sequencing suffers from imperfect sensitivity, especially for CIC::DUX4.

Renal CIC-LEUTX rearranged sarcoma with multiple pulmonary metastases: a case report and literature review.

BACKGROUND: CIC-rearranged sarcomas (CRS) are a group of heterogeneous tumors which mostly occur in the soft tissues of limbs and trunk, and are highly invasive with poor prognosis. Here, we describe a rare case of CRS that occurred in the left kidney with a CIC-LEUTX rearrangement. CASE PRESENTATION: A 45-year-old male was admitted to hospital with a dry cough for more than two months without obvious cause. Physical examination and laboratory tests revealed no notable abnormality. The CT scan demonstrated a mass in the left kidney and multiple nodules in both lungs. The percutaneous core needle biopsy showed similar histomorphology and immunophenotype of small round cell malignant tumors. Genetic test revealed a CIC-LEUTX gene fusion. CONCLUSIONS: We present a rare primary renal CRS with multiple pulmonary metastases, and LEUTX is confirmed as the fusion partner of CIC gene for the first time in a renal case.

A Practical Approach to Small Round Cell Tumors Involving the Gastrointestinal Tract and Abdomen.

Small round cell neoplasms are diagnostically challenging owing to their clinical and pathologic overlap, necessitating use of large immunopanels and molecular analysis. Ewing sarcomas (ES) are the most common, but EWSR1 is translocated in several diverse neoplasms, some with round cell morphology. Molecular advances enable classification of many tumors previously termed 'atypical ES'. The current WHO Classification includes two new undifferentiated round cell sarcomas (with CIC or BCOR alterations), and a group of sarcomas in which EWSR1 partners with non-Ewing family transcription factor genes. This article reviews the spectrum of small round cell sarcomas within the gastrointestinal tract and abdomen.

[Clinical and morphological characteristics of Ewing's sarcoma and the algorithm for diagnosing undifferentiated round cell sarcomas]. / Kliniko-morfologicheskaya kharakteristika sarkomy Yuinga i algoritm diagnostiki nedifferentsirovannykh kruglokletochnykh sarkom.

BACKGROUND: The group of undifferentiated round cell sarcomas, according to the World Health Organization Classification, in addition to Ewing's sarcoma (ES), includes round cell sarcoma with rearrangement of the EWSR1 gene with partners not from the ETS gene family, sarcoma with BCOR gene alterations, CIC -rearranged sarcoma. Despite the fact that all tumors have clear histological and immunological criteria, their diagnosis can be difficult, given the fact that there are overlapping variants of the morphological picture and immunophenotype both within the group and with other round cell tumors. OBJECTIVE: Present a comparative analysis of genetically verified ES, sarcoma with BCOR gene alterations and CIC-rearranged sarcoma. MATERIAL AND METHODS: A comparative study of biopsy specimens of bones, soft tissues and internal organs was carried out in 118 patients with ES, 10 with BCOR gene alterations and 8 with CIC-rearranged sarcomas. All cases were genetically verified. The following research methods were used: histological, immunohistochemical, RT-PCR, RNA sequencing and FISH. RESULTS: Within our cohort, it was shown that ES predominantly affects bones, while soft tissue localization is more typical for the other two undifferentiated round cell sarcomas. Histologically, in the overwhelming majority of cases, ES is characterized by a monomorphic round-cell structure; on the contrary, heterogeneous structure is typical for sarcoma with alterations of the BCOR gene, CIC-rearranged sarcoma. High sensitivity and specificity of CD99/NKX2.2 co-expression for ES, BCOR/SATB2/TLE1 for sarcoma with BCOR gene alterations, high specificity and low sensitivity of WT1/ETV4 co-expression for CIC-rearranged sarcoma was shown. CONCLUSION: For the differential diagnosis of undifferentiated round-cell sarcomas, it is necessary to take into account the clinical, morphology when compared with the data of the IHC study, and verification by molecular genetic methods is necessary to improve the accuracy of diagnosis.

[CIC-rearranged sarcoma: a case report and literature review]. / CIC-perestroennaya sarkoma: klinicheskoe nablyudenie i obzor literatury.

CIC-rearranged sarcoma is a rare and extremely aggressive tumor that occurs mainly in soft tissues. Despite the fact that identification of a characteristic genetic rearrangement is necessary to verify the diagnosis, in most cases, the correct diagnosis can be made by comparing histological signs and a characteristic immunophenotype, which greatly speeds up the diagnosis. The article describes a case of CIC-rearranged sarcoma in a 14-year girl with the successful application of the CWS-2009 treatment protocol.

Clinicopathological features of five cases of CIC::DUX4 positive sarcomas, including literature review.

According to the recent World Health Organization (WHO) classification, CIC-rearranged sarcomas, including CIC::DUX4-positive sarcomas constitute an aggressive subtype of undifferentiated round cell sarcomas. There is a single study on these tumors from our subcontinent. We present clinicopathological features of 5 additional cases of this tumor entity, including literature review. Thirty-nine undifferentiated round cell sarcomas, excluding Ewing sarcomas (ES), were tested for CIC::DUX4 fusion, including Type I (165 base pair size) and II (230 bp) by reverse transcription-polymerase chain reaction. Twenty-five of those tumors were tested for EWSR1 gene rearrangement, 5 for SS18 and 4 for SS18::SSX fusion, and were negative for those tests. Five tumors (12.8 %) were positive for CIC::DUX4(Type II) fusion. Five CIC:: DUX4-positive sarcomas occurred in 4 males and one female; of 25-43 years of age, in soft tissues, including thigh (n = 2), chest wall (n = 1), iliac region (n = 1) and foot (n = 1). Tumor size varied from 2.2 to 19 cm. Microscopically, the tumors were predominantly composed of nodules and sheets of malignant round to epithelioid cells, including "rhabdoid-like" (n = 2) and spindle-shaped (n = 2) with eosinophilic to vacuolated cytoplasm (4/5), distinct nucleoli (4/5), brisk mitoses, focal myxoid to hyalinised stroma (4/5) and necrosis (5/5). Immunohistochemically, tumor cells were positive for WT1 (5/5), calretinin (3/4), pan-keratin (1/4), CD99/MIC2 ("dot-like" to cytoplasmic membranous) (4/4), while negative for desmin (0/4), S100P (0/4), and NKX2.2 (0/5). INI1/SMARCB1 was retained (3/3). All patients underwent excision with adjuvant radiotherapy and chemotherapy (Ewing sarcoma regimen). A single patient developed recurrence, and 2 developed pulmonary metastasis, including one with brain metastasis. CIC:: DUX4-positive sarcomas are ultra-rare tumors, that mainly occur in the soft tissues and in young adult patients. Histopathologically, these tumors display a wide spectrum, including round to epithelioid cells, variable amount of cytoplasmic vacuolization and myxoid stroma with necrosis. Immunohistochemically, these tumors express WT1 and calretinin. Despite adjuvant therapies, these tumors have dismal outcomes, especially in large-sized tumors. CIC::DUX4-positive sarcomas need to be differentiated from their histopathological mimics, including ES, in view of significant treatment-related implications.

CIC-rearranged sarcoma presenting with superior vena cava syndrome: case report.

CIC-rearranged sarcomas are rare mesenchymal neoplasms belonging to the family of undifferentiated small round cell sarcomas. This report details the case of a 45-year-old man presenting with symptoms of mediastinal compression, radiological diagnosis of a mediastinal mass and rapid evolution to full-blown superior vena cava syndrome. The emergency was successfully managed with a pharmacological approach. Formulation of a pathological diagnosis of CIC-rearranged sarcoma was initially supported by fluorescence in situ hybridisation findings and later validated by next-generation sequencing, which showed CIC-DUX4 gene fusion. A chemotherapy regimen was started with immediate benefits for the patient. The spectrum of pathological entities able to cause superior vena cava syndrome is wide, and recognition of rare causes is important to tailor the therapeutic approach to the specific disease. This is, to the best of our knowledge, the first report of CIC-rearranged sarcoma presenting with superior vena cava syndrome.

A global collaboRAtive study of CIC-rearranged, BCOR::CCNB3-rearranged and other ultra-rare unclassified undifferentiated small round cell sarcomas (GRACefUl).

BACKGROUND: Undifferentiated small round cell sarcomas (URCSs) represent a diagnostic challenge, and their optimal treatment is unknown. We aimed to define the clinical characteristics, treatment, and outcome of URCS patients. METHODS: URCS patients treated from 1983 to 2019 at 21 worldwide sarcoma reference centres were retrospectively identified. Based on molecular assessment, cases were classified as follows: (1) CIC-rearranged round cell sarcomas, (2) BCOR::CCNB3-rearranged round cell sarcomas, (3) unclassified URCSs. Treatment, prognostic factors and outcome were reviewed. RESULTS: In total, 148 patients were identified [88/148 (60%) CIC-rearranged sarcoma (median age 32 years, range 7-78), 33/148 (22%) BCOR::CCNB3-rearranged (median age 17 years, range 5-91), and 27/148 (18%) unclassified URCSs (median age 37 years, range 4-70)]. One hundred-one (68.2%) cases presented with localised disease; 47 (31.8%) had metastases at diagnosis. Male prevalence, younger age, bone primary site, and a low rate of synchronous metastases were observed in BCOR::CCNB3-rearranged cases. Local treatment was surgery in 67/148 (45%) patients, and surgery + radiotherapy in 52/148 (35%). Chemotherapy was given to 122/148 (82%) patients. At a 42.7-month median follow-up, the 3-year overall survival (OS) was 92.2% (95% CI 71.5-98.0) in BCOR::CCNB3 patients, 39.6% (95% CI 27.7-51.3) in CIC-rearranged sarcomas, and 78.7% in unclassified URCSs (95% CI 56.1-90.6; p < 0.0001). CONCLUSIONS: This study is the largest conducted in URCS and confirms major differences in outcomes between URCS subtypes. A full molecular assessment should be undertaken when a diagnosis of URCS is suspected. Prospective studies are needed to better define the optimal treatment strategy in each URCS subtype.

Expanding the Molecular Diversity of CIC-Rearranged Sarcomas With Novel and Very Rare Partners.

Capicua transcriptional repressor (CIC)-rearranged sarcoma represents a distinct pathologic entity and constitutes the second most prevalent category of undifferentiated round cell sarcomas (URCSs) after Ewing sarcoma. The 2 most common translocations are t(4;19) and t(10;19), resulting in CIC fusions with either DUX4 and DUX4L paralog, respectively; however, other rare variant fusions have also been reported. In this study, we expand the molecular spectrum of CIC-gene partners, reporting on 5 cases of URCSs showing CIC fusions with AXL, CITED1, SYK, and LEUTX by targeted RNA or DNA sequencing. There were 4 female patients and 1 male patient with a wide age range (12-70 years; median, 36 years). Four cases occurred in the deep soft tissues (lower extremity, 3; neck, 1) and 1 case in the central nervous system (midbrain/thalamus). All cases showed similar histologic findings within the spectrum of URCSs. Immunohistochemistry, showed variable positivity for ETV4 in 4 of the 4 cases and positive results for ERG in 3 of the 4 cases and for WT1 in 1 of the 4 cases. CD31 showed positivity in 2 of the 3 cases, including one coexpressing ERG. Unsupervised clustering of methylation profiles by T-distributed stochastic neighborhood embedding performed in 4 cases showed that all clustered tightly together and along the CIC sarcoma methylation class. RNA-sequencing data showed consistent upregulation of ETV1 and ETV4 mRNA in all cases examined, at similar levels to CIC::DUX4 URCSs. Our study expands the molecular diversity of CIC-rearranged URCSs to include novel and rare partners, providing morphologic, immunohistochemical, gene expression, and methylation evidence supporting their classification within the family of tumors harboring the more common DUX4/DUX4L partner genes.

Approach to Primary Vertebral Tumors in the Light of the 2020 Updated World Health Organization Classification of Bone Tumors.

AIM: To define a new approach for primary vertebral tumors by discussing them in the light of 2020 World Health Organization (WHO) classification of bone tumors. MATERIAL AND METHODS: In this study, we have discussed primary vertebral tumors in light of the 2020 Updated WHO Classification of Soft Tissue and Bone Tumors. RESULTS: Chondroblastoma and chondromyxoid fibroma has been classified in the benign category, while synovial chondromatosis has been moved from the benign category to the intermediate category. In the updated classification, grade I chondrosarcoma has been classified in the malignant category with grade II?III chondrosarcomas. Minor amendments have been made for osteosarcoma subtypes. Neoplasms of undetermined origin, such as aneurysmal cysts, simple bone cysts, fibrosis dysplasia, and osteofibrous dysplasia, have been classified as neoplastic lesions. Chordomas have been classified into ?not otherwise stated,? poorly differentiated chordomas, and dedifferentiated chordomas. Ewing?s sarcomas have been classified in a separate section for undifferentiated, small, round cell sarcomas of the bone and soft tissue. In this section, three distinct subsets different from Ewing?s sarcoma have been discussed. CIC-rearranged sarcoma, BCOR-rearranged sarcoma, and round cell sarcomas with EWSR1 gene fusion with non-ETS family members. CONCLUSION: In this study, we have reviewed the new classifications and discussed their effect on decision making in spinal oncologic surgery.

Patterns of care and outcome of CIC-rearranged sarcoma patients: A nationwide study of the French sarcoma group.

BACKGROUND: CIC-rearranged sarcomas (CIC-RS) represent the most frequent subset of "Ewing-like" undifferentiated small round cell sarcomas. These tumors tend to be more aggressive than Ewing sarcomas. Moreover, treatment strategy can differ according to teams. The primary aim of this retrospective study was to describe the characteristics, treatments, and outcome for patients with CIC-RS included in the French NETSARC+ database. METHODS: Pediatric and adult patients from 13 French centers with a diagnosis of CIC-RS were registered from October 2008 to March 2021. Patients and tumors characteristics were collected from the national network NETSARC+ database (http://netsarc.sarcomabcb.org). CIC-RS diagnosis was pathologically and molecularly confirmed with a central review by expert pathologists. Two groups of patients were studied: those treated as classical Ewing sarcomas (cohort EwS) and those treated as high-grade soft tissue sarcomas (cohort STS) according to ESMO and/or EpSSG guidelines. Survival was calculated using the Kaplan-Meier method and the log-rank test was used to compare survival. RESULTS: Among 79 patients, the male/female sex ratio was 0.7 and the median age at diagnosis was 27 years (range 2-87). With a median follow-up of 37 months, 39 patients died of the disease. Median overall survival from diagnosis was 18 months, with no significant difference between both cohorts (p = 0.9). Nevertheless, when focusing on patients with metastatic disease at diagnosis (N = 21), all patients from cohort STS died of disease while some patients from cohort EwS were still alive and in complete remission. CONCLUSION: FSG experience confirms the aggressive clinical course of CDS patients regardless of chemotherapy regimen.

Molecular and immunohistochemical testing of bone tumours: review and update.

Primary bone tumours can pose diagnostic problems due to their overlapping radiologic and histologic features. Given the recent advancement in our understanding of the biology of bone tumours, multiple immunohistochemical and molecular markers have been devised to aid in their diagnosis. This review provides brief updates on select bone tumours, including chondrosarcomas, benign chondrogenic tumours, osteosarcomas, benign osteogenic tumours, fibroosseous lesions, vascular tumours, osteoclastic giant cell-rich or cystic tumours, chordoma, adamantinoma, small round blue cell sarcomas, and others. We discuss their salient molecular features and novel immunohistochemical correlates, along with some tips to avoid common diagnostic pitfalls.