PD-1 combined with TRBC1 and pan-T cell antibodies for robustly monitoring angioimmunoblastic T-cell lymphoma

Abstract

Background: The diagnosis of AITL is challenging. It may be delayed or even missed due to critical clinical conditions and its histologic and immunophenotypic overlap with other neoplastic and reactive lymphoid proliferations.

Objective: The key objective is to obtain an efficient diagnosis, sensitive disease monitoring and treatment efficacy assessment of AITL using multiparameter flow cytometry (MFC).

Methods: In total, 167 de novo AITL patients were immunophenotypically profiled using sensitive MFC. We precisely identified the aberrant T-cell populations of AITL and performed an in-depth description of their phenotypic characteristics in comparison with their residual normal CD4+ T cells. A comparison of Programmed death receptor-1 (PD-1) expression was performed among AITL and other T-cell lymphomas.

Results: MFC detected a neoplastic T-cell population in 94.1% (80/85) of tissue, 71.5% (108/151) of bone marrow (BM), 100% (8/8) of peripheral blood (PB) and 78.6% (11/14) of body fluid samples. The most frequent immunophenotypic aberrations included the absence and diminished expression of CD3 (71.25% in tissues, 71.3% in BM, 75% in PB, 81.8% in hydrothorax and ascites specimens), followed by the loss or partial loss of CD7 (71.25% in LN, 67.6% in BM, 50% in PB, 81.8% in hydrothorax and ascites specimens). The immunophenotyping of neoplastic T-cell populations showed a high degree of similarity among different sites of the same patient and they might change over time but were relatively stable. Bright PD-1 expression showed high sensitivity and specificity in differentiating AITL from other T-cell lymphomas. In 14 AITL patients, neoplastic T-cell populations were initially missed by T-cell screening tube but were successfully discovered by bright PD-1 expression.

Conclusion: T-cell screening tube can reliably screen neoplastic T-cell populations in AITL patients with typical immunophenotyping, such as loss of surface CD3 and loss of CD7 with a relatively high ratio. Bright PD-1 expression is essential for identifying aberrant T cells in almost all AITLs. The clonality assessment antibody TRBC1 is efficient for robustly and cheaply assessing T-cell clonality. Using PD-1 and TRBC1 combined with pan-T cell antibodies can make a precise diagnosis of AITL and also sensitively monitor minimal residual disease regardless of the antigenic drift of the neoplastic T cells.

Keywords: PD-1; TRBC1; angioimmunoblastic T-cell lymphoma; immunophenotyping; multiparameter flow cytometry; pan-T antibodies.

Figure 1

The expression features of pan-T cell antigens of AITL patients in tissue (A) and bone marrow (B). (A) Compared with the corresponding non-neoplastic CD4+ T cells in AITL, neoplastic T cells had bright CD2 expression and diminished CD3, CD4, and CD5 expression. CD7 mean fluorescent intensity (MFI) had no significant difference between CD7+ neoplastic T cells and normal CD4+ T cells. (B) In bone marrow samples, neoplastic T cells had obviously diminished CD3, CD4, and CD5 expression, and there were no significant difference of CD2 expression between neoplastic T cells and normal CD4+ T cells. CD7 mean fluorescent intensity (MFI) had no significant difference between CD7-positive neoplastic T cells and normal CD4+ T cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

The most common aberrancies of pan-T cell antigens in AITL. (A) PB specimen in which neoplastic CD4+ T cells had bright CD45 and PD-1 expression, and diminished CD4 expression, lack of CD3, CD7, and TRBC1 with slightly larger side scatter. The same immunophenotyping was observed in BM, PB, and LN specimens. (B) FNA sample in which CD4+ aberrant T cells had bright CD4, CD2, and PD-1 expression, CD200 was positive, CD5, and CD45 were diminished; CD3, CD7, and TRBC1 were absent; Side scatter was enlarged.

Figure 3

Flow cytometric findings of PD-1 expression in non-neoplastic CD4+ T cells and T-cell lymphoma. (A) PD-1 mean fluorescent intensity (MFI) in bone marrow samples. The corresponding non-neoplastic CD4+ T cells in AITL were used as a control. The PD-1 MFI in AITL is significantly higher than that in control T cells and other T-cell lymphomas. (B) ROC curve of PD-1 MFI bone marrow samples shows high sensitivity and specificity in differentiating AITL from non-AITL T-cell lymphoma (p < 0.001, AUC 0.970: sensitivity 100.0% and specificity 83.33% for a cutoff of MFI 1162). (C) PD-1 mean fluorescent intensity (MFI) in tissue samples. The PD-1 MFI in AITL is significantly higher than that in control T cells and other T-cell lymphomas. (D) ROC curve of PD-1 MFI tissue samples shows high sensitivity and specificity in differentiating AITL from non-AITL T-cell lymphoma (p < 0.001, AUC 0.971: sensitivity 91.67% and specificity 93.75% for a cutoff of MFI 2023). ***p < 0.001.

Figure 4

Flow cytometric findings in non-T cell lymphoma. (A) CD4+ T cells with CD10 and bright PD-1 expression were observed in follicular lymphoma. (B) CD4+ T cells with CD200 and bright PD-1 expression were seen in tuberculous granuloma. (C) Expansion of CD4+T cells with bright PD-1 expression was present in nodular lymphocyte-predominant Hodgkin lymphoma. (D) CD4+ T cells with distinctive bright PD-1 expression were observed in reactive follicular hyperplasia. TRBC1 was polyclonal in these populations.

Figure 5

Some AITL immunophenotyping could not be detected by the T-cell screen tube. (A,D,E,F) show BM specimens. (B,C) show tissue samples. (A) Aberrant T cells were CD3+, CD4+, and CD10+ with bright PD-1 expression, and CD7 was partially expressed. (B) Neoplastic T cells were CD4+, CD7+, and CD10+ with slightly diminished CD3 and CD5 expression and bright PD-1 expression. (C) Tumor cells were CD3+, CD5+, CD7+, and CD200+ with diminished CD4 expression and bright CD2, CD56, and PD-1 expression, and CD10 was negative (not shown). (D) Abnormal T cells were CD3+, CD4+, CD5+, and CD200+ with bright CD7 and PD-1 expression, and CD10 was negative. (E) An extremely low ratio of CD7 negative T cells was CD3+, CD4+, and CD10+ with bright CD5 and PD-1 expression. The immunophenotyping of neoplastic T cells shifted over time (E,F) before and after therapy, respectively. TRBC1 did not show polyclonal expression in these populations.