CIC Rearrangement Research Articles

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 (ESs) are the most common SBRCSs, defined by gene fusions involving EWSR1 and transcription factors of the ETS gene family, while the most frequent non-EWSR1-rearranged SBRCSs harbor a CIC rearrangement. Unfortunately, the identification of CIC::DUX4 translocation events, the most common CIC rearrangement, is challenging with current methods. Here, a machine-learning approach to support SBRCS diagnosis relying on gene expression profiles measured via targeted sequencing is presented. The analyses on a curated cohort of 69 soft tissue tumors (STT) showed markedly distinct expression patterns for SBRCS subgroups. A random forest (RF) classifier trained on ES and CIC-rearranged cases predicted probabilities of being CIC-rearranged > 0.9 for CIC-rearranged-like sarcomas and < 0.6 for other SBRCS. Testing on a retrospective cohort of 1335 routine diagnostic cases identified 15 candidate CIC-rearranged tumors with a probability > 0.75, all of which supported by expert histopathological reassessment. Furthermore, the multi-gene RF classifier appeared advantageous over using high ETV4 expression alone, previously proposed as surrogate to identify CIC rearrangement. Taken together, the expression-based classifier can offer valuable support for SBRCS pathological diagnosis.

134. Novel fusions in aggressive infant sarcomas: Expanding the scope of 'CIC-rearranged' sarcoma without CIC rearrangement

CIC-rearranged sarcomas are newly defined undifferentiated soft tissue tumors with CIC-associated fusions, mostly with DUX4, and dismal prognosis. CIC fusions activate PEA3 family genes, ETV1/4/5, leading to tumorigenesis and progression. We report three pediatric sarcomas with novel fusions and similar gene-expression/methylation patterns without CIC rearrangement. One disseminated tumor of unknown origin and two high-grade CNS tumors of unclear histological diagnosis were received for genomic evaluation. All three patients were infants with aggressive diseases, and two experienced rapid disease deterioration and death. A series of IHC staining and comprehensive NGS panel analyses for mutations, copy number alterations, and fusions were not diagnostic. Whole-transcriptome sequencing identified an ATXN1L-NUTM2A fusion in case 1 and ATXN1-NUTM2A in the two CNS tumors. ETV1/4/5 and WT1 overexpression were observed in all three cases. Methylation analyses predicted CIC-rearranged sarcoma for all cases. Retrospective IHC staining on case 2 demonstrated ETV4 and WT1 overexpression. An ATXN1-DUX4 fusion was recently reported in a CNS sarcoma that showed a gene expression pattern similar to that of CIC-rearranged sarcomas. ATXN1 and ATXN1L interact with CIC forming a transcription repressor complex. We propose that ATXN1/ATXN1L-associated fusions disrupt their interaction with CIC and decrease the transcription repressor complex, leading to downstream gene overexpression. These three cases, including a non-CNS tumor, with novel ATXN1/ATXN1L-associated fusions and features of CIC-rearranged sarcomas further expand the scope of 'CIC-rearranged' sarcomas without CIC rearrangement. Additional cases are needed to demonstrate if ATXN1/ATXN1L-NUTM2A fusions are associated with younger age and more aggressive diseases.

Primary retroperitoneal perirenal CIC rearrangement sarcoma: A case report.

Primary retroperitoneal perirenal CIC rearrangement sarcoma is rare. The current case report presents a 69-year-old male patient with this pathology, including the clinical features, pathomorphology and immunohistochemistry, and CIC gene rupture detected by fluorescence in situ hybridization (FISH). Furthermore, the relevant literature was reviewed. Histologically, the tumor was composed of diffuse nests of small- to medium-sized juvenile round blue cells with hyperchromatic nuclei, prominent nucleoli and occasional mitotic signs. The tumor involved adipose tissue with no obvious hemorrhagic necrotic foci. Immunohistochemistry indicated scattered expression of CD99 in tumor cells. FISH examination suggested that the CIC gene was fragmented and translocated.

Novel ATXN1/ATXN1L::NUTM2A fusions identified in aggressive infant sarcomas with gene expression and methylation patterns similar to CIC-rearranged sarcoma

CIC-rearranged sarcomas are newly defined undifferentiated soft tissue tumors with CIC-associated fusions, and dismal prognosis. CIC fusions activate PEA3 family genes, ETV1/4/5, leading to tumorigenesis and progression. We report two high-grade CNS sarcomas of unclear histological diagnosis and one disseminated tumor of unknown origin with novel fusions and similar gene-expression/methylation patterns without CIC rearrangement. All three patients were infants with aggressive diseases, and two experienced rapid disease deterioration and death. Whole-transcriptome sequencing identified an ATXN1-NUTM2A fusion in the two CNS tumors and an ATXN1L-NUTM2A fusion in case 3. ETV1/4/5 and WT1 overexpression were observed in all three cases. Methylation analyses predicted CIC-rearranged sarcoma for all cases. Retrospective IHC staining on case 2 demonstrated ETV4 and WT1 overexpression. ATXN1 and ATXN1L interact with CIC forming a transcription repressor complex. We propose that ATXN1/ATXN1L-associated fusions disrupt their interaction with CIC and decrease the transcription repressor complex, leading to downstream PEA3 family gene overexpression. These three cases with novel ATXN1/ATXN1L-associated fusions and features of CIC-rearranged sarcomas may further expand the scope of “CIC-rearranged” sarcomas to include non-CIC rearrangements. Additional cases are needed to demonstrate if ATXN1/ATXN1L-NUTM2A fusions are associated with younger age and more aggressive diseases.

A Primary Capicua Transcriptional Repressor (CIC)-Rearranged Round Cell Sarcoma of the Neck: A Case Report and Literature Review

This is a case report of a CIC-rearranged sarcoma located in the neck. Our patient was a 36 year-old-woman with unremarkable past medical history who presented with a left lateralized neck mass evolving for one year without other signs. The physical examination showed a bulky left cervical mass of 12cm long axis, with infected and necrotic areas. There was no palpable lymph nodes. A computed tomography (CT) scan was performed and confirmed the suspicious nature of the lesion and did not show distant metastasis. Histology (completed with Fluorescence in situ Hybridization (FISH) analysis) of a biopsy confirmed the diagnosis of round cell sarcoma with CIC rearrangement. As the tumor was unresectable, we started with chemotherapy but the tumor progressed after three cycles. The patient could not receive a second line chemotherapy because of the deterioration of the general condition and died after 1 month. In conclusion, CIC-rearranged sarcoma is a rare tumor and has a poorer prognosis than the classic Ewing Sarcoma (ES). Its treatment is still challenging. More research is needed to establish the optimal treatment strategies.

Reassessment of EWSR1 gene rearrangement-negative undifferentiated round cell sarcomas in bone and soft tissues by fluorescence in situ hybridization

Objective: To unravel the CIC rearrangement sarcomas and BCOR-CCNB3 sarcomas from EWSR1 rearrangement-negative undifferentiated round cell sarcomas in the bone and soft tissues. Methods: Twenty-eight cases of EWSR1 rearrangement-negative undifferentiated round cell sarcomas of bone and soft tissues, tested for CIC rearrangement and BCOR rearrangement by fluorescence in situ hybridization and related immunostaining were analyzed, and some of the BCOR rearrangement cases were verified by reverse transcription-polymerase chain reaction. Results: Five of 28 (17.9%) tested cases were positive for CIC rearrangement and six (21.4%) for BCOR rearrangement. Histopathologically, CIC rearrangement sarcomas comprised nodular aggregates of round to polygonal cells, containing hyperchromatic nuclei, prominent nucleoli and moderate cytoplasm, with focal variable necrosis and myxoid stroma. BCOR-CCNB3 sarcomas mostly comprised diffusely arranged, round to oval to short spindly cells with angulated nuclei, vesicular chromatin, inconspicuous nucleoli and interspersed vessels. Immunohistochemically, five of six BCOR-CCNB3 sarcomas showed CCNB3 immunostaining, which could be helpful for diagnosis. Two patients with CIC rearrangement sarcoma died of the diseases in seven months and twenty-two months. One patient with BCOR-CCNB3 sarcoma died of the diseases in forty-six months. Conclusions: Overall, 39.3% of the EWSR1 rearrangement-negative undifferentiated round cell sarcomas are CIC rearrangement sarcomas and BCOR-CCNB3 sarcomas. Molecular testing is helpful for diagnosis.

Superficial sarcomas with CIC rearrangement are aggressive neoplasms: A series of eight cases.

CIC rearranged sarcomas have significant overlap with Ewing sarcoma, are aggressive, and typically present in deep soft tissue. They most commonly have a t(4;19)(q35;q13) with CIC-DUX4 fusion. Superficial presentation is rare. We report eight (6F, 2M; median 45-years-old, range 14-65) superficial CIC-rearranged sarcomas, involving the extremities (n = 4), vulva (n = 2), and trunk (n = 2). The tumors were composed of nodules/sheets of round cells with necrosis and hemorrhage separated by dense hyaline bands. Tumor cells had vesicular chromatin, prominent nucleoli and frequent mitotic figures. One showed pagetoid spread. Targeted next-generation sequencing was positive for CIC-DUX4 fusion (6/6); fluorescence in situ hybridization (FISH) was positive for CIC rearrangement (2/3). Eight of eight had evidence of CIC-DUX4 fusion/rearrangement by molecular techniques. Immunohistochemistry was positive for CD99+ (8/8) and DUX4+ (4/4). FISH for EWSR1 rearrangement was negative (5/5). Of five patients with at least 6 months follow-up, three of five died of disease, all within 2 years of presentation. One is alive with disease at 48 months. One is disease free at 3 months. Superficial CIC-rearranged sarcomas should be considered in cases exhibiting features reminiscent of Ewing sarcoma, but with increased pleomorphism and/or geographic necrosis. In contrast to superficial Ewing sarcomas, superficial CIC-rearranged sarcomas are aggressive.

DNA methylation profiling distinguishes Ewing-like sarcoma with EWSR1-NFATc2 fusion from Ewing sarcoma.

Recent studies revealed divergent gene expression patterns in Ewing sarcoma (EwS) with canonical EWSR1-ETS gene fusions and undifferentiated round cell sarcomas (URCS) with EWSR1 rearrangements fused to the non-ETS gene NFATc2. Thus, the question arises whether the latter tumors really belong to EwS. We collected five cases matching the group of URCS with EWSR1-NFATc2 fusion and performed DNA methylation and copy number profiling. Results were compared to methylation data of 30 EwS with various EWSR1-ETS fusions and one EwS with FUS-ERG fusion, 16 URCS with CIC rearrangement and 10 URCS with BCOR alteration and a total of 81 EWSR1-associated soft tissue sarcomas including 7 angiomatoid fibrous histiocytomas, 7 clear cell sarcomas of the soft tissue, 28 desmoplastic small round cell tumors, 10 extraskeletal myxoid chondrosarcomas and 29 myxoid liposarcomas. Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis of DNA methylation data revealed a homogeneous methylation cluster for URCS with EWSR1-NFATc2 fusion, which clearly segregated from EwS and the other subtypes. Copy number profiles of EWSR1-NFATc2 cases showed recurrent losses on chromosome 9q and segmental gains on 20q13 and 22q12 involving the EWSR1 and NFATc2 loci, respectively. In summary, URCS with EWSR1-NFATc2 fusion share a distinct DNA methylation signature and carry characteristic copy number alterations, which emphasizes that these sarcomas should be considered separately from EwS.

Round cell sarcoma of the colon with CIC rearrangement

BackgroundThe CIC-rearranged sarcoma is a very rare highly aggressive malignant soft tissue group of tumors. It has recently been described as highly aggressive soft tissue tumors of children and young adults sharing similar morphological features with the Ewing sarcoma. The digestive localization is exceptional.Case presentationA 14-year-old male presented with a history of abdominal pain for 1 year, which increased in intensity over the last 2 months. Imaging findings showed a large heterogeneous mesenteric mass on the left flank of the abdomen. Exploratory laparotomy was performed and revealed a large cystic hypervascularized mass depending on the transverse colon and mesocolon. A wide excision of the lesion was performed with segmental colectomy. No postoperative complications were noted. The microscopic examination revealed a vaguely nodular growth of undifferentiated small round cells, arranged in solid sheets separated by thin fibrous septa with a scarce stroma. After an uncomplicated post-operative course, the patient was referred for chemotherapy. The patient died 2 months later with a peritoneal and pleural progression.ConclusionsThe CIC-rearranged sarcoma is an aggressive tumor. There is no standard therapy for this rare disease. Their treatment includes surgery and chemotherapy. Resistance to chemotherapy is common. Further publications and studies will help to determine a standard therapy for this rare disease.

CIC break-apart fluorescence in-situ hybridization misses a subset of CIC-DUX4 sarcomas: a clinicopathological and molecular study.

Approximately 60-70% of high-grade round-cell sarcomas that lack the Ewing sarcoma breakpoint region 1 (EWSR1) rearrangement harbour a rearrangement of the CIC gene, most commonly CIC-DUX4. Recent studies have established that CIC-rearranged sarcomas constitute a distinct group characterized by recognizable histology and immunoprofiles, such as positivity for ETV4 and WT1 and negativity for NKX2.2. Although these sarcomas are diagnosed increasingly in practice by fluorescence in-situ hybridization (FISH) with CIC break-apart probes, the optimal modality to diagnose these sarcomas has not been determined. In this study, we describe four round-cell sarcomas that showed false-negative results by CIC break-apart FISH assays. These sarcomas showed characteristic histology of CIC-rearranged sarcomas, and all were immunohistochemically positive for ETV4 and WT1 and negative for NKX2.2. Although FISH showed non-atypical negative signals for CIC rearrangement, high-throughput RNA sequencing identified CIC-DUX4 and its fusion breakpoint in all cases. Their clinical and histological findings, as well as fusion points determined by RNA sequencing, did not differ significantly from those of nine FISH-positive CIC-DUX4 sarcoma cases. We estimated that the FISH false-negative rate for CIC-rearranged sarcomas was 14%. Although neither histology nor immunoprofiles (e.g. ETV4 and WT1) are entirely sensitive or specific for CIC-rearranged sarcomas, the observation that these four cases were identified successfully by such phenotypes suggested their practical utility. CIC break-apart FISH assays missed a significant minority of CIC-DUX4 sarcomas, and full awareness of typical morphology and judicious immunohistochemical work-ups, including analyses of ETV4 and WT1, should complement diagnostic assessment.

The utility of ETV1, ETV4 and ETV5 RNA in-situ hybridization in the diagnosis of CIC-DUX sarcomas.

A recently characterized group of undifferentiated small round cell sarcomas harbours fusions of the genes CIC and DUX4. Studies report a distinctive gene expression profile for these sarcomas, including expression of E26 transformation-specific (ETS) family proto-oncogenic transcription factors ETV1, ETV4 and ETV5. To test the utility of an ancillary diagnostic technique for these tumours, we evaluated chromogenic RNA in-situ hybridization assays for ETV1, ETV4 and ETV5 as diagnostic adjuncts for this emerging group of highly malignant sarcomas. We tested six confirmed CIC-DUX4 sarcomas and 105 lesions in the differential, including 48 Ewing sarcomas for expression of ETV1, ETV4 and ETV5, scoring expression utilizing a previously validated scale. ETV1 and ETV4 were positive in five of six cases, while ETV5 was positive in six of six. No Ewing sarcoma or other sarcoma tested showed coexpression of these transcripts, while one ETV1/ETV4/ETV5 triple positive previously unclassified round cell sarcoma was identified as harbouring a CIC rearrangement by break-apart fluorescence in-situ hybridization (FISH). We identified overexpression of ETV1, ETV4 and ETV5 transcripts in situ in CIC-DUX4 sarcomas using a robust assay in routine archival sections. One previously unclassified round cell sarcoma showed ETV1/4/5 positivity, and was proved to harbour a CIC rearrangement by break-apart FISH. The sensitivity and specificity observed with our in-situ hybridization assay implies potential utility as an ancillary diagnostic technique, particularly when faced with limited biopsy samples.

Recurrent CIC Gene Abnormalities in Angiosarcomas: A Molecular Study of 120 Cases With Concurrent Investigation of PLCG1, KDR, MYC, and FLT4 Gene Alterations.

Angiosarcoma (AS) is a rare sarcoma subtype showing considerable clinicopathologic and genetic heterogeneity. Most radiation-induced AS show MYC gene amplifications, with a subset of cases harboring KDR, PTPRB, and PLCG1 mutations. Despite recent advances, the genetic abnormalities of most primary AS remain undefined. Whole-transcriptome sequencing was initiated in 2 index cases of primary soft tissue AS with epithelioid morphology occurring in young adults for novel gene discovery. The candidate abnormalities were validated and then screened by targeted sequencing and fluorescence in situ hybridization in a large cohort of 120 well-characterized AS cases. Findings were subsequently correlated with the status of KDR, PLCG1, MYC, and FLT4 gene abnormalities. The clinicopathologic relevance and prognostic significance of these genetic changes were analyzed by statistical methods. Concurrent CIC mutations and CIC rearrangements were identified in both index cases, with a CIC-LEUTX fusion detected in 1 case. Upon screening, an additional visceral AS in a young adult had a complex CIC rearrangement, whereas 6 others harbored only CIC mutations. All 3 CIC-rearranged AS cases lacked vasoformation and had a solid growth of round, epithelioid to rhabdoid cells, showing immunoreactivity for CD31 and Ets-related gene and sharing a transcriptional signature with other round cell sarcomas, including CIC-rearranged tumors. Overall, CIC abnormalities occurred in 9% (9/98) of cases, affecting younger patients with primary AS, with an inferior disease-free survival. In contrast, PLCG1 and KDR mutations occurred in both primary and secondary AS cases, accounting for 9.5% and 7%, respectively, with a predilection for breast and bone/viscera location, regardless of MYC status. MYC amplification was present in most secondary AS related to breast cancer (91%) compared with other causes (25%) or primary AS (7%). FLT4-amplified AS lacked PLCG1/KDR mutations, occurring predominantly in MYC-amplified population, and showed poor prognosis.

Undifferentiated Small Round Cell Sarcoma With t(4;19)(q35;q13.1) CIC-DUX4 Fusion

A subset of small round cell sarcomas remains difficult to classify. Among these, a rare tumor harboring a t(4;19)(q35;q13.1) with CIC-DUX4 fusion has been described. The aim of this study is to better understand its clinicopathologic features. Four cases of CIC-DUX4 sarcoma, all arising in adults (3 women, 1 man, aged 20 to 43 y), were identified using conventional cytogenetic, reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) methods. All 4 tumors demonstrated CIC-DUX4 fusion transcript by both RT-PCR and FISH and CIC rearrangement by FISH. Cytogenetic results from 2 tumors showed t(4;19)(q35;q13.1) occurring as part of a simple karyotype in 1 tumor and as part of a complex karyotype in the other, the latter from a postchemotherapy specimen. Both tumors harbored trisomy 8 and lacked any other known sarcoma-associated translocation. No EWS or SYT rearrangements were detected by RT-PCR or FISH. The tumors had small round cell morphology with a distinctive constellation of histologic features including extensive geographic necrosis, mild nuclear pleomorphism with coarse chromatin and prominent nucleoli, clear cell areas, and focal myxoid matrix. Only focal staining for CD99 was present in each tumor. Two had very focal cytokeratin staining. All tumors were negative for desmin, myogenin, TLE-1, and S100 protein, whereas nuclear INI-1 staining was retained. The tumors were highly aggressive, and all patients died of disseminated disease within 16.8 months. CIC-DUX4 sarcoma represents a novel translocation-associated sarcoma with distinctive histopathologic features and rapid disease progression.