The Discovery of a Novel Gene Fusion in Sarcoma
Unraveling the molecular mystery of undifferentiated small round cell sarcoma through the identification of CRTC1-SS18 gene fusion
When a 12-year-old girl arrived at the hospital with abdominal pain, doctors discovered a mysterious mass in her retroperitoneal space. The tumor displayed an unusual combination of calcification and fatty components, initially mistaken for a benign teratoma. Only after surgical removal and advanced genetic testing did the true diagnosis emerge: undifferentiated small round cell sarcoma (USRCS)—a rare and complex cancer that continues to puzzle oncologists worldwide 2 .
USRCS tumors contain complex genetic alterations that make diagnosis and treatment challenging.
Pathologists struggle to distinguish between various USRCS subtypes using conventional methods.
This case exemplifies the diagnostic challenges posed by USRCS, a group of highly aggressive malignancies affecting children and young adults. These tumors arise in bones or soft tissues, composed of primitive "small round cells" that seem to defy classification. For decades, pathologists struggled to distinguish between various USRCS subtypes, until groundbreaking genetic research uncovered a novel CRTC1-SS18 gene fusion—discovery that has reshaped our understanding of sarcoma biology and opened new avenues for targeted therapies 3 6 .
Undifferentiated small round cell sarcomas represent a diverse family of malignant tumors characterized by their small, blue-staining cells under the microscope. The latest World Health Organization classification recognizes four main categories within this group, each defined by distinct genetic alterations that drive their development 9 .
The most established member, predominantly driven by fusions between the EWSR1 gene and ETS family transcription factors like FLI1 or ERG. These account for approximately 85-90% of Ewing cases and result in potent oncogenic transcription factors that disrupt normal cellular programming 5 .
Defined primarily by CIC-DUX4 fusions, these aggressive tumors often present in soft tissues of the head, neck, or trunk. They typically express characteristic markers like WT1 and ETV4, with a strong tendency for rapid progression and poor response to conventional chemotherapy 2 5 .
This category includes tumors with BCOR-CCNB3 fusions or other BCOR genetic alterations. Unlike other USRCS, these tend to affect bones more frequently than soft tissues and account for approximately 4% of round cell sarcomas, often appearing in the pelvis or lower limbs 2 .
This group encompasses tumors with fusions involving EWSR1 and non-ETS partners like NFATc2 or PATZ1. NFATc2-related tumors show a preference for bone rather than soft tissues, while PATZ1-fusion sarcomas strongly favor the thoracoabdominal region 2 .
The groundbreaking identification of the novel CRTC1-SS18 gene fusion was published in The Journal of Pathology in 2018, representing the culmination of sophisticated genetic detective work. Researchers employed high-throughput RNA sequencing (RNA-seq) to analyze undifferentiated small round cell sarcomas that lacked any known translocation, focusing particularly on those with morphological features similar to Ewing sarcoma but without the characteristic EWSR1-ETS fusions 3 6 .
The research team discovered that the CRTC1-SS18 fusion joins exons 1-2 of CRTC1 on chromosome 19 with either exon 2 or 4 of SS18 on chromosome 18. CRTC1 (CREB Regulated Transcription Coactivator 1) normally functions as a regulator of CREB-mediated transcription, while SS18 (SS18 Subunit of BAF Chromatin Remodeling Complex) plays a role in chromatin remodeling. Their fusion creates a chimeric oncoprotein that likely disrupts normal transcriptional programs in susceptible cells, driving malignant transformation 1 3 .
What made this discovery particularly compelling was the observation that tumors with CRTC1-SS18 fusion demonstrated elevated expression of NTRK1, a gene encoding a receptor tyrosine kinase that represents a promising therapeutic target. This finding suggested that CRTC1-SS18 sarcomas might be vulnerable to emerging TRK inhibitor drugs, potentially offering new treatment options for affected patients 3 .
The identification and validation of the CRTC1-SS18 fusion followed a meticulous, multi-step approach that combined state-of-the-art genomic technologies with classical molecular biology techniques. The experimental journey began with RNA sequencing of tumor samples from two different cancer centers, both representing undifferentiated small round cell sarcomas that had defied conventional classification 3 .
Researchers obtained tumor samples with appropriate ethical approvals and patient consent. They extracted total RNA from both fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) tissue, carefully quantifying RNA quality and quantity using advanced instrumentation like the Agilent 2100 Bioanalyzer 3 .
The team prepared cDNA libraries using Illumina's TruSeq RNA Sample Prep Kit, followed by paired-end sequencing on Illumina platforms (HiSeq 2000 for fresh tissue; NextSeq 500 for FFPE samples). This generated comprehensive transcriptome data capable of detecting novel fusion events 3 .
Researchers employed multiple computational tools—FusionCatcher, STAR-Fusion, and FusionMap—to identify potential gene fusions from the RNA-seq data. This multi-algorithm approach enhanced the reliability of findings by cross-validating results across different methodologies 3 .
The putative CRTC1-SS18 fusion was confirmed using several orthogonal methods:
To demonstrate the oncogenic potential of CRTC1-SS18, researchers introduced the fusion gene into recipient cells and observed resulting phenotypic changes. These expression experiments revealed that cells carrying the fusion became morphologically distinct and demonstrated enhanced oncogenic properties compared to control cells 3 .
The gene expression patterns of CRTC1-SS18 tumors were compared against other known sarcoma subtypes using clustering analyses. Remarkably, the CRTC1-SS18 cases grouped closely with EWSR1-CREB1-positive tumors, suggesting potential biological relationships or common downstream pathways 3 .
| Validation Method | Key Finding | Significance |
|---|---|---|
| RNA Sequencing | Identical CRTC1-SS18 fusion in two independent cases | Supported recurrence rather than random translocation event |
| RT-PCR | Successful amplification of predicted fusion transcript | Confirmed fusion at the RNA level |
| FISH | SS18 gene rearrangement in tumor cells | Validated chromosomal breakpoint at DNA level |
| Functional Assays | Enhanced oncogenic potential in fusion-expressing cells | Established biological relevance in tumor development |
| Reagent/Technology | Primary Function | Application in CRTC1-SS18 Discovery |
|---|---|---|
| RNA Sequencing | Comprehensive transcriptome analysis | Initial discovery of novel CRTC1-SS18 fusion |
| TruSeq RNA Sample Prep Kit | Library preparation for sequencing | Generated cDNA libraries compatible with Illumina platforms |
| SS18 Break-Apart FISH Probe | Visualize chromosomal rearrangements | Validated SS18 gene breakage in tumor nuclei |
| FusionCatcher Software | Computational fusion detection | Identified candidate fusions from RNA-seq data |
| NTRK1 Antibody (ab76291) | Detect NTRK1 protein expression | Revealed elevated NTRK1 in CRTC1-SS18 tumors |
The investigation of rare sarcomas requires increasingly sophisticated approaches. Recent efforts have established patient-derived tumor organoids (tumoroids) as powerful models for studying these malignancies. These three-dimensional cultures maintain the histological features, genetic alterations, and cellular heterogeneity of original tumors, enabling more physiologically relevant drug testing and biological studies 5 .
For proteomic characterization, researchers now employ mass spectrometry-based approaches that can quantify thousands of proteins from minimal biopsy material. This technology has demonstrated that different USRCS subtypes exhibit distinct protein expression signatures, which could lead to improved diagnostic classification when genetic testing is inconclusive .
Since the initial discovery, additional cases of CRTC1-SS18 sarcoma have been identified, providing a clearer clinical profile. A 2024 study analyzing six total cases reported a male-to-female ratio of 1:2 with a median age of 34 years (range: 12-42 years). These tumors primarily occur in intramuscular locations of the lower extremity and display characteristic histologic features including uniform round-to-epithelioid cells with moderate eosinophilic cytoplasm arranged in sheets and nests within a prominent desmoplastic stroma 1 4 .
Immunohistochemical findings have proven somewhat variable and nonspecific, with patchy expression of CD99 and variable staining for ALK, GATA3, and cyclin D1. This heterogeneity complicates diagnosis based on morphology and immunohistochemistry alone, emphasizing the essential role of molecular testing for accurate classification 1 .
The clinical course of CRTC1-SS18 sarcomas appears notably variable. While one initially reported case demonstrated aggressive behavior with a fatal outcome, two others followed a relatively indolent course with gradual growth over 6-7 years before resection. Among the documented cases, two developed metastatic disease—one to bilateral lungs and another to locoregional lymph nodes 1 4 .
This variability in clinical behavior underscores the need for further research to identify factors predicting aggressiveness.
The discovery of elevated NTRK1 expression in CRTC1-SS18 tumors suggests potential vulnerability to TRK inhibitors.
| Clinical Feature | Pattern | Clinical Significance |
|---|---|---|
| Age Distribution | Median 34 years (range 12-42) | Affects predominantly young adults |
| Gender Preference | Female predominance (2:1 ratio) | Contrasts with some other sarcoma subtypes |
| Tumor Location | Predominantly lower extremity intramuscular | Guides radiographic evaluation |
| Clinical Behavior | Variable (indolent to aggressive) | Complicates treatment decision-making |
| Metastatic Pattern | Lungs, lymph nodes | Informs surveillance strategies |
The identification of CRTC1-SS16 as a recurrent fusion in undifferentiated small round cell sarcoma represents more than just the addition of another subtype to the classification scheme—it exemplifies the transformative power of modern genomics to refine cancer diagnosis and treatment. As sequencing technologies become more accessible and comprehensive, we can anticipate further dissection of rare cancers into molecularly defined entities with tailored therapeutic approaches.
The ongoing establishment of patient-derived tumoroid biobanks containing various SRCS subtypes promises to accelerate drug discovery and personalized treatment approaches 5 .
Proteomic characterization continues to complement genomic findings by revealing subtype-specific protein signatures and activated pathways that might be targeted therapeutically .