American journal of translational research · 2025
Integrating flow cytometric profile and gene mutation analysis of intact lymph nodes to refine lymphoma immunophenotype.
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Peng Qun-Yi, Chen Hong-Yuan, Zheng Yan, Zhu Xiong-Peng, Li Chun-Tuan
Аннотация
OBJECTIVE: To evaluate the diagnostic use of integrating multi-parameter flow cytometry (FCM), histopathology, and gene mutation analysis for lymphoma classification using intact lymph node (LN) samples.
METHODS: Intact LN samples from 109 patients with lymphadenopathy were retrospectively analyzed by pathology, multi-color FCM, and next-generation sequencing (NGS) targeting 62 lymphoma-related genes.
RESULTS: FCM immunophenotyping showed high concordance with pathology (56/56 lymphoma cases detected by FCM were pathologically confirmed). PD-1 on T cells was significantly elevated in B-cell lymphoma (BCL), especially diffuse large BCL (DLBCL) (P<0.05). All BCLs exhibited monotypic intracellular cKappa or cLambda expression. Significant differences in cell size (FSC) were observed: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) (mean FSC: 89.42±6.01) and Follicular Lymphoma cells (FL; 93.88±4.94) were smaller than normal B-cells (102.09±11.58), while DLBCL cells (121.84±9.17) were larger (all P<0.05). Subtypes showed distinct mutation profiles, including IGHV (9/11) in CLL/SLL; BCL2 (5/7) and EZH2 (4/7) in FL; and BCL6 (5/13) in DLBCL. Mutation-guided FCM confirmed BCL2 protein expression in two FL cases and BCL6 in one DLBCL case. T/NK-cell lymphomas showed aberrant antigen expression and restricted TRBC1 clonality (1.17%±1.61% or >96.4%) outside the normal polyclonal range (36.60%±7.21%).
CONCLUSION: FCM on intact LNs is a robust tool with high pathologic concordance. Integrating genetic mutation data with FCM provides a powerful, multi-parameter strategy. This approach moves beyond standard immunophenotyping to include mutation-associated antigens, thereby refining lymphoma classification and enhancing diagnostic accuracy.
METHODS: Intact LN samples from 109 patients with lymphadenopathy were retrospectively analyzed by pathology, multi-color FCM, and next-generation sequencing (NGS) targeting 62 lymphoma-related genes.
RESULTS: FCM immunophenotyping showed high concordance with pathology (56/56 lymphoma cases detected by FCM were pathologically confirmed). PD-1 on T cells was significantly elevated in B-cell lymphoma (BCL), especially diffuse large BCL (DLBCL) (P<0.05). All BCLs exhibited monotypic intracellular cKappa or cLambda expression. Significant differences in cell size (FSC) were observed: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) (mean FSC: 89.42±6.01) and Follicular Lymphoma cells (FL; 93.88±4.94) were smaller than normal B-cells (102.09±11.58), while DLBCL cells (121.84±9.17) were larger (all P<0.05). Subtypes showed distinct mutation profiles, including IGHV (9/11) in CLL/SLL; BCL2 (5/7) and EZH2 (4/7) in FL; and BCL6 (5/13) in DLBCL. Mutation-guided FCM confirmed BCL2 protein expression in two FL cases and BCL6 in one DLBCL case. T/NK-cell lymphomas showed aberrant antigen expression and restricted TRBC1 clonality (1.17%±1.61% or >96.4%) outside the normal polyclonal range (36.60%±7.21%).
CONCLUSION: FCM on intact LNs is a robust tool with high pathologic concordance. Integrating genetic mutation data with FCM provides a powerful, multi-parameter strategy. This approach moves beyond standard immunophenotyping to include mutation-associated antigens, thereby refining lymphoma classification and enhancing diagnostic accuracy.