Diagnostic and Treatment Approach for CD3 and CD20 Positive Immunohistochemistry
Critical Diagnostic Interpretation
The simultaneous presence of CD3 and CD20 positivity requires immediate expanded immunohistochemical analysis to distinguish between a reactive lymph node with mixed T and B cell populations, composite lymphoma, or rare lymphoma subtypes with aberrant marker expression. 1, 2
The key diagnostic challenge is that CD3 typically marks T-cells while CD20 marks B-cells—co-expression in the same cells is uncommon and demands careful evaluation. 1, 3 In most cases, this pattern represents either:
- Reactive lymph node: CD3 stains T-cells in T-cell zones and CD20 stains B-cells in B-cell follicles, creating a diffuse mixed pattern 2, 3
- Composite lymphoma: Two distinct lymphoma populations present simultaneously 2
- Aberrant marker expression: Rare cases where lymphoma cells express markers from both lineages 4
Mandatory Expanded Immunohistochemistry Panel
Order the following additional markers immediately to establish definitive lineage and diagnosis: 1, 2
B-cell markers:
- CD79a (more reliable than CD20, especially post-rituximab or with plasmacytic differentiation) 3, 5
- PAX5 6, 5
- BCL-2 (to detect follicular lymphoma translocation) 2, 3
- BCL-6 2
T-cell markers:
- CD5 2, 5
- CD4 and CD8 (to determine T-cell subset) 2, 4
- CD43 and CD45RO (for peripheral T-cell lymphomas) 5
Additional essential markers:
- CD30 and CD15 (to rule out Hodgkin lymphoma, where Reed-Sternberg cells are typically CD3-negative and CD20-positive in <40% of cases) 6, 1
- Ki-67 (proliferation index to assess aggressiveness) 2, 3
- CD10 and MUM1 (for cell-of-origin classification) 2
Molecular Studies Required
If immunohistochemistry shows ambiguous or conflicting results, molecular analysis is mandatory: 4
- PCR for T-cell receptor (TCR) gene rearrangement 4, 7
- PCR for immunoglobulin heavy chain (IgH) gene rearrangement 4, 7
- Gene expression profiling if available (though remains investigational) 6
A case report documented primary CNS T-cell lymphoma with aberrant CD20 and CD79a expression, where molecular studies showing clonal TCR rearrangements were essential for correct diagnosis. 4
Treatment Algorithm Based on Final Diagnosis
If CD20-Positive B-Cell Lymphoma (Most Likely Scenario):
Administer R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) every 21 days for 6-8 cycles as the standard treatment for diffuse large B-cell lymphoma. 6, 8
- Rituximab is FDA-approved for CD20-positive B-cell NHL in combination with CHOP or other anthracycline-based regimens 8
- Confirm CD20 positivity on expanded panel to ensure rituximab eligibility 6
- Premedicate before each rituximab infusion to prevent infusion reactions 8
- Screen for hepatitis B (HBsAg and anti-HBc) before initiating rituximab due to reactivation risk 8
Stratify treatment intensity by age and International Prognostic Index (IPI): 6
- Young patients (age <60) with low-intermediate risk (aaIPI ≤1): R-CHOP × 6-8 cycles every 21 days 6
- Young patients with high-intermediate/high risk (aaIPI ≥2): R-CHOP × 6-8 cycles; consider dose-dense regimens (every 14 days) and CNS prophylaxis 6
- Elderly patients (age 60-80): R-CHOP × 8 cycles every 21 days, or 6 cycles if given every 14 days 6
If Classical Hodgkin Lymphoma:
Treat with ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) or other Hodgkin-specific regimens, NOT R-CHOP. 6
- Reed-Sternberg cells express CD30 (100%) and CD15 (most cases) but are typically CD3-negative and CD45-negative 6, 1
- CD20 is positive in <40% of Hodgkin cases 1
- Rituximab is NOT indicated for classical Hodgkin lymphoma 6
If T-Cell Lymphoma with Aberrant CD20:
Treat with T-cell lymphoma-directed chemotherapy (CHOP without rituximab or alternative regimens); consider adding rituximab only if CD20 expression is confirmed on neoplastic cells. 6, 4
- One case report showed PCNSTCL with aberrant CD20/CD79a expression treated with vincristine, methotrexate, and rituximab, though the patient died within one month 4
- Standard CHOP remains the backbone for peripheral T-cell lymphomas 6
Critical Pre-Treatment Workup
Complete the following staging before initiating therapy: 6, 1
- CT chest/abdomen/pelvis 6
- PET/CT scan (strongly recommended for accurate staging and response assessment) 6, 1
- Bone marrow aspirate and biopsy 6
- Complete blood count with differential 6, 8
- Comprehensive metabolic panel including LDH and uric acid 6
- HIV, hepatitis B (HBsAg, anti-HBc), and hepatitis C screening 6, 8
- Cardiac function assessment (LVEF) before anthracycline therapy 6, 1
- Calculate IPI score for prognostication 6, 1
Tumor Lysis Syndrome Prevention
For patients with high tumor burden, initiate prophylaxis before chemotherapy: 6
- Prednisone 100 mg orally for several days as "prephase" treatment 6
- Aggressive hydration 9
- Allopurinol or rasburicase 9
- Monitor electrolytes closely 9
CNS Prophylaxis Considerations
Administer intrathecal chemotherapy (methotrexate or cytarabine) for high-risk patients: 6, 9
- High-intermediate or high IPI score (≥2 adverse factors) 6
- More than one extranodal site 6
- Bone marrow involvement 6
- Testicular, paranasal sinus, or epidural involvement 6
- Elevated LDH 6
Common Diagnostic Pitfalls to Avoid
- Do not misinterpret reactive T-cells (CD3+) admixed with neoplastic B-cells (CD20+) as composite lymphoma—this is the most common error 2
- Do not rely on CD20 alone for B-cell lineage determination—CD79a is more reliable, especially after rituximab therapy or with plasmacytic differentiation 3, 5
- Do not assume CD20 positivity automatically means B-cell lymphoma—rare T-cell lymphomas can aberrantly express CD20 4
- Do not use fine-needle aspiration alone—excisional lymph node biopsy provides adequate tissue for comprehensive immunohistochemistry and molecular studies 6
- Heterogeneous CD20 staining patterns in B-cell lymphomas (46.4% of cases) may still indicate bone marrow infiltration—do not dismiss as negative 10
Response Assessment Strategy
Repeat imaging after 3-4 cycles and at treatment completion: 6, 1