How should I request a sarcoma next‑generation sequencing (NGS) test?

How to Request Sarcoma NGS Testing

Request both DNA-based and RNA-based NGS panels simultaneously at diagnosis or when considering systemic therapy, with RNA sequencing being essential for fusion detection in sarcomas. 1

Testing Strategy

Panel Selection

• RNA-based NGS is mandatory for sarcoma evaluation because it identifies fusion transcripts (the functional product of chromosomal rearrangements) and provides information on fusion partners and functionality 1
• RNA sequencing overcomes the critical limitation of DNA-only panels, which can miss fusion breakpoints occurring in large intronic regions or complex genomic sequences 1
• Request panels covering at least 138-200 genes that include both fusion detection and point mutation analysis 2, 3

Specific Genes/Alterations to Ensure Coverage

Your NGS request should explicitly cover:

Fusion genes (diagnostic and therapeutic targets):

• EWSR1 fusions (FLI1, ERG, WT1, POU5F1, ATF1)
• SS18 (SYT) fusions
• NTRK1/2/3 fusions (actionable)
• ALK, ROS1, RET fusions (actionable)
• FGFR1/2/3 fusions (actionable)
• COL1A1-PDGFB, NAB2-STAT6, PAX3-MAML3
• CIC-DUX4, BCOR-CCNB3
• FUS-DDIT3, ETV6-NTRK3 1, 2, 3

Point mutations and copy number variations:

• MDM2 and CDK4 amplifications (liposarcoma/osteosarcoma)
• TP53, RB1, NF1 mutations
• CDKN2A/B deletions
• KIT, PIK3CA mutations (potentially actionable)
• BRAF V600E (actionable)
• SMARCB1, BCOR alterations 4, 3

Timing of Testing

Order NGS early in the disease course - ideally at initial diagnosis or before first-line systemic therapy. The evidence shows that 73% of patients received NGS late in their disease course, which limited therapeutic options 4. Early testing enables:

• Diagnostic refinement (impacts 41.1% of cases) 2
• Treatment planning before clinical deterioration
• Clinical trial enrollment opportunities

Clinical Scenarios Where NGS is Essential

High Priority (Order Immediately):

1. Round cell sarcomas with atypical morphology - NGS identifies diagnostic fusions in 87.5% and changes treatment in 18.7% 2
2. Undifferentiated sarcomas - NGS detects fusions in 21.1%, reclassifies all positive cases, and alters treatment in 75% 2
3. Histologically ambiguous tumors where morphology and immunohistochemistry are inconclusive 5, 3
4. Advanced disease requiring systemic therapy - actionable alterations found in 33-42% of cases 4

Standard Practice:

• All soft tissue sarcomas being considered for systemic therapy
• Bone sarcomas with unusual features or requiring treatment decisions

Specimen Requirements

• Fresh frozen tissue is preferred for RNA-based NGS (RNA degrades in formalin-fixed tissue)
• If only FFPE available, ensure adequate tumor cellularity and tissue quality
• Coordinate with pathology early to preserve appropriate specimens 5

What to Include in Your Request

Write your NGS order to specify:

1. "RNA-based fusion panel AND DNA-based mutation panel" (not just "NGS")
2. Include tumor mutational burden (TMB) and microsatellite instability (MSI) assessment
3. Request specific coverage of NTRK, ALK, ROS1, RET, FGFR fusions (these are FDA-approved targets)
4. Note clinical context: "for diagnostic clarification and identification of actionable targets"

Common Pitfalls to Avoid

• Don't order DNA-only panels - you will miss most diagnostic fusions in sarcomas 1
• Don't wait until multiple lines of therapy have failed - 67% of patients who didn't receive NGS-based treatment had no actionable targets, but late testing meant clinical deterioration prevented treatment even when targets existed 4
• Don't assume negative results mean no fusions - if breakpoints fall outside panel coverage, results may be false negative 1
• Don't skip MDM2 assessment - while RNA panels may not detect MDM2 amplification directly, request supplementary FISH or note that ≥3 fusions in 12q13-15 region or MDM2 expression ≥100 TPM suggests dedifferentiated liposarcoma 6

Expected Outcomes

Based on the evidence, NGS will:

• Provide clinically informative results in 50% of cases 2
• Enable diagnostic reclassification in 41% 2
• Identify actionable alterations in 33-42% 4
• Change treatment decisions in 26% 2
• Improve progression-free survival when NGS-directed therapy is available (9 vs 2 months, p=0.023) 4

Integration with Multidisciplinary Care

Submit cases to a molecular tumor board (MTB) for interpretation, as genomic findings require integration with histopathology and clinical context for optimal treatment decisions 5. This is particularly important because 79% of sarcomas have low TMB and are microsatellite stable, making immunotherapy unlikely to work despite occasional high TMB cases 4.