What is the role of CD99, Fli-1 (Friend leukemia integration 1), and Nkx2.2 in the immunohistochemistry (IHC) diagnosis of Ewing's sarcoma?

Immunohistochemical Markers in Ewing's Sarcoma Diagnosis: CD99, FLI-1, and NKX2.2

CD99, FLI-1, and NKX2.2 form a powerful diagnostic panel for Ewing's sarcoma, with the combination of CD99 and NKX2.2 offering the highest specificity (98%) for accurate diagnosis and differentiation from other small round cell tumors. 1

CD99 (MIC2 Gene Product)

CD99 is the cornerstone immunohistochemical marker for Ewing's sarcoma diagnosis:

• Expressed in nearly all cases of Ewing's sarcoma
• Shows characteristic strong membranous staining pattern
• Corresponds to the expression of the MIC2 gene 2
• While sensitive, CD99 alone lacks specificity as it can be positive in other small round cell tumors

FLI-1 (Friend Leukemia Integration 1)

FLI-1 serves as a complementary marker in the diagnostic panel:

• Nuclear transcription factor commonly expressed in Ewing's sarcoma
• Directly related to the most common translocation in Ewing's sarcoma, t(11;22)(q24;q12)
• This translocation creates the EWS-FLI1 fusion protein found in approximately 85-90% of cases 2
• While useful, FLI-1 has limitations in specificity as it can also be expressed in lymphoblastic lymphomas and certain vascular tumors

NKX2.2

NKX2.2 is a newer and highly valuable addition to the diagnostic panel:

• Homeodomain-containing transcription factor involved in neuroendocrine/glial differentiation
• Direct transcriptional target of the EWS-FLI1 fusion protein 3
• Shows nuclear staining pattern
• Sensitivity of 80-93% for Ewing's sarcoma 3, 1, 4
• Specificity of 84-89% when used alone 3, 1
• Present in 93% of genetically confirmed Ewing's sarcomas with typically diffuse (>50%) and moderate-to-strong staining intensity 3

Diagnostic Value of Combined Markers

The true power of these markers emerges when used in combination:

• CD99 + NKX2.2: This combination achieves 98% specificity for Ewing's sarcoma diagnosis 1
• Only rare cases of mesenchymal chondrosarcoma and small cell carcinoma may show positivity for both markers 1, 5
• The combination effectively distinguishes Ewing's sarcoma from other small round cell tumors, particularly CIC-DUX4 and BCOR-CCNB3 sarcomas 4, 5

Important Diagnostic Pitfalls

When using these markers, be aware of these potential pitfalls:

1. False positives for NKX2.2:

   • Olfactory neuroblastomas (80%)
   • Mesenchymal chondrosarcomas (75%)
   • Rare cases of small cell carcinomas, neuroblastomas, and synovial sarcomas 3, 5

2. Decalcification effects:

   • Bone specimens may show reduced immunoreactivity due to decalcification processes 4
   • Consider this when interpreting negative results in bone-derived specimens

3. Confirmatory molecular testing:

   • While immunohistochemistry is valuable, detection of characteristic translocations (particularly t(11;22)(q24;q12)) by cytogenetics or PCR remains the gold standard for definitive diagnosis 2
   • Present in >90% of Ewing's sarcoma cases

Recommended Diagnostic Algorithm

For optimal diagnosis of Ewing's sarcoma:

1. Start with morphologic assessment (small round blue cell tumor)
2. Apply CD99 immunostaining (strong membranous pattern)
3. Add NKX2.2 immunostaining (nuclear pattern)
4. If both are positive, high likelihood of Ewing's sarcoma
5. Consider FLI-1 as additional supporting evidence
6. Confirm with molecular testing for EWSR1 rearrangements when available

This approach ensures the most accurate diagnosis, which is critical for proper treatment planning and ultimately impacts patient survival and quality of life.