Comparison of the composition of lymphocyte subpopulations in non-relapse and relapse patients with squamous cell carcinoma of the head and neck before, during radiochemotherapy and in the follow-up period: a multicenter prospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG)

Minli Niu, Stephanie E Combs, Annett Linge, Mechthild Krause, Michael Baumann, Fabian Lohaus, Nadja Ebert, Ingeborg Tinhofer, Volker Budach, Jens von der Grün, Franz Rödel, Anca-Ligia Grosu, Gabriele Multhoff, Minli Niu, Stephanie E Combs, Annett Linge, Mechthild Krause, Michael Baumann, Fabian Lohaus, Nadja Ebert, Ingeborg Tinhofer, Volker Budach, Jens von der Grün, Franz Rödel, Anca-Ligia Grosu, Gabriele Multhoff

Abstract

Background: Radiochemotherapy (RCT) has been shown to induce changes in immune cell homeostasis which might affect antitumor immune responses. In the present study, we aimed to compare the composition and kinetics of major lymphocyte subsets in the periphery of patients with non-locoregional recurrent (n = 23) and locoregional recurrent (n = 9) squamous cell carcinoma of the head and neck (SCCHN) upon primary RCT.

Methods: EDTA-blood of non-locoregional recurrent SCCHN patients was collected before (t0), after application of 20-30 Gy (t1), in the follow-up period 3 (t2) and 6 months (t3) after RCT. In patients with locoregional recurrence blood samples were taken at t0, t1, t2 and at the time of recurrence (t5). EDTA-blood of age-related, healthy volunteers (n = 22) served as a control (Ctrl). Major lymphocyte subpopulations were phenotyped by multiparameter flow cytometry.

Results: Patients with non-recurrent SCCHN had significantly lower proportions of CD19+ B cells compared to healthy individuals before start of any therapy (t0) that dropped further until 3 months after RCT (t2), but reached initial levels 6 months after RCT (t3). The proportion of CD3+ T and CD3+/CD4+ T helper cells continuously decreased between t0 and t3, whereas that of CD8+ cytotoxic T cells and CD3+/CD56+ NK-like T cells (NKT) gradually increased in the same period of time in non-recurrent patients. The percentage of CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) decreased directly after RCT, but increased above initial levels in the follow-up period 3 (t2) and 6 (t3) months after RCT. Patients with locoregional recurrence showed similar trends with respect to B, T cells and Tregs between t0 and t5. CD4+ T helper cells remained stably low between t0 and t5 in patients with locoregional recurrence compared to Ctrl. NKT/NK cell subsets (CD56+/CD69+, CD3-/CD56+, CD3-/CD94+, CD3-/NKG2D+, CD3-/NKp30+, CD3-/NKp46+) increased continuously up to 6 months after RCT (t0-t3) in patients without locoregional recurrence, whereas in patients with locoregional recurrence, these subsets remained stably low until time of recurrence (t5).

Conclusion: Monitoring the kinetics of lymphocyte subpopulations especially activatory NK cells before and after RCT might provide a clue with respect to the development of an early locoregional recurrence in patients with SCCHN. However, studies with larger patient cohorts are needed.

Trial registration: Observational Study on Biomarkers in Head and Neck Cancer (HNprädBio), NCT02059668. Registered on 11 February 2014, https://ichgcp.net/clinical-trials-registry/NCT02059668 .

Keywords: Immunophenotyping; Lymphocyte subpopulations; NK cell subsets; Prediction of locoregional recurrence; Radiochemotherapy; SCCHN.

Conflict of interest statement

No competing interest exists by any of the authors.

© 2021. The Author(s).

Figures

Fig. 1
Fig. 1
Immunophenotyping of major lymphocyte subpopulations. Percentages of a CD3−/CD19+ B cells, b CD3+ T cells, c CD3+/CD4+ T helper cells, d CD3+/CD8+ cytotoxic T cells, e CD4+/CD25+/FoxP3+ Tregs, f CD3+/CD56+ NK-like T cells in healthy controls (Ctrl, n = 22), non-recurrent (n = 23) and recurrent patients (n = 9) with SCCHN before (t0), after application of 20–30 Gy (t1), 3 months (t2), 6 months (t3) after treatment and at time of locoregional recurrence (t5, 3–15 months after t0). The data show mean values ± standard deviation of the percentage of positively stained cells. Significances are illustrated between t0 and other time points (tx) after start of RCT as well as between controls (Ctrl) and all time points of RCT (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)
Fig. 1
Fig. 1
Immunophenotyping of major lymphocyte subpopulations. Percentages of a CD3−/CD19+ B cells, b CD3+ T cells, c CD3+/CD4+ T helper cells, d CD3+/CD8+ cytotoxic T cells, e CD4+/CD25+/FoxP3+ Tregs, f CD3+/CD56+ NK-like T cells in healthy controls (Ctrl, n = 22), non-recurrent (n = 23) and recurrent patients (n = 9) with SCCHN before (t0), after application of 20–30 Gy (t1), 3 months (t2), 6 months (t3) after treatment and at time of locoregional recurrence (t5, 3–15 months after t0). The data show mean values ± standard deviation of the percentage of positively stained cells. Significances are illustrated between t0 and other time points (tx) after start of RCT as well as between controls (Ctrl) and all time points of RCT (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)
Fig. 2
Fig. 2
Immunophenotyping of NK cell subpopulations. Percentages of a CD56+/CD69+ NKT/NK cells, b CD3−/CD56+ NK cells, c CD3−/CD94+ NK cells, d CD3−/NKG2D+ NK cells, e CD3−/NKp30+ NK cells, f CD3−/NKp46+ NK cells in healthy controls (Ctrl, n = 22), non-recurrent (n = 23) and recurrent patients (n = 9) with SCCHN before (t0), after application of 20–30 Gy (t1), 3 months (t2), 6 months (t3) after treatment and at time of locoregional recurrence (t5, 3–15 months after t0). The data show mean values ± standard deviation of the percentage of positively stained cells. Significances are illustrated between t0 and other time points (tx) after start of RCT as well as between controls (Ctrl) and all time points of RCT (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)
Fig. 2
Fig. 2
Immunophenotyping of NK cell subpopulations. Percentages of a CD56+/CD69+ NKT/NK cells, b CD3−/CD56+ NK cells, c CD3−/CD94+ NK cells, d CD3−/NKG2D+ NK cells, e CD3−/NKp30+ NK cells, f CD3−/NKp46+ NK cells in healthy controls (Ctrl, n = 22), non-recurrent (n = 23) and recurrent patients (n = 9) with SCCHN before (t0), after application of 20–30 Gy (t1), 3 months (t2), 6 months (t3) after treatment and at time of locoregional recurrence (t5, 3–15 months after t0). The data show mean values ± standard deviation of the percentage of positively stained cells. Significances are illustrated between t0 and other time points (tx) after start of RCT as well as between controls (Ctrl) and all time points of RCT (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

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