Utility of LymphoTrack for clonality testing in formalinfixed, paraffin-embedded tissue: integrated panel- and specimen-level comparison with fragment analysis

Background: Next-generation sequencing-based clonality assays provide sequence-level resolution but remain vulnerable to panel-specific failure in formalin-fixed, paraffin-embedded (FFPE) tissue. We evaluated LymphoTrack performance and integrated multi-panel interpretation. Methods: Seventeen FFPE specimens from 16 patients underwent 51 immunoglobulin heavy-chain (IGH) and 12 T-cell receptor (TCR) panel runs. Results were classified as Positive, Negative, or Fail and compared with lineage-level fragment-analysis interpretations. Panels targeting each lineage were integrated into specimen-level interpretations. Quality-control metrics associated with panel failure were explored. Results: Among 51 IGH panel runs, 37 (72.5%) were concordant, four (7.8%) discordant, and 10 (19.6%) Fail; all Fail results occurred in FR1 or FR2. IGH specimen-level agreement was 16/17 (94.1%; 95% confidence interval [CI], 73.0% to 99.0%). LymphoTrack classified four IGH specimens as monoclonal and 13 as polyclonal, including six based on incomplete panel coverage. One such specimen (FR1/FR2 Fail and FR3 Negative) was discordant with monoclonal fragment analysis. TCR specimen-level agreement was 5/6 (83.3%; 95% CI, 43.6% to 97.0%). In the discordant TCR specimen, T-cell receptor gamma (TRG) fragment analysis was oligoclonal, whereas both LymphoTrack TRG and T-cell receptor beta were Positive. A post-polymerase chain reaction library concentration threshold of ≤4.5 ng/μL identified all Fail panels but had limited specificity (58.5%) and precision (31.3%). Conclusions: LymphoTrack showed high specimen-level agreement with fragment analysis despite panel-specific technical vulnerability in FFPE tissue. Partial panel failure had asymmetric implications: a Positive panel preserved a monoclonal interpretation, whereas polyclonal results with incomplete panel coverage remained vulnerable to discordance. Reporting incomplete panel coverage may help communicate this limitation.