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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)

Comparison of the composition of lymphocyte subpopulations in non-relapse and relapse patients... 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 volun‑ teers (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 *Correspondence: minli.niu@tum.de Center for Translational Cancer Research ( TranslaTUM), Radiation Immuno‑Oncology Group, Klinikum rechts der isar, TU München ( TUM), Einsteinstr. 25, 81675 Munich, Germany Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Niu et al. Radiat Oncol (2021) 16:141 Page 2 of 12 + + + 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:// clini caltr ials. gov/ ct2/ show/ NCT02 059668. Keywords: SCCHN, Prediction of locoregional recurrence, Immunophenotyping, Radiochemotherapy, Lymphocyte subpopulations, NK cell subsets Background oro- and hypopharynx at UICC stadium III or IV with- Head and neck cancer is the seventh most common can- out any remote metastases were included into the cer worldwide with an incidence rate of approximately study  (Table  1). The patients are a subset of patients 600.000 new cases in 2012 [1]. Despite progress in radia- recruited into the HNprädBio study (clinicaltrials. tion oncology over the past decades, the 5  year survival gov, NCT02059668) of the German Cancer Consor- rate of 40–60% of patients with locally advanced SCCHN tium Radiation Oncology Group (DKTK-ROG), which is still not satisfying [1]. In advanced inoperable stages, is based on the availability of blood samples in the time SCCHN patients are treated primarily with RCT. Poten- period from May 2014 till December 2016. Nine of these tial prognostic markers for the response to RCT include patients developed a locoregional recurrence within a the human papillomavirus (HPV) infection status, tumor median time period of 11  months (range: 3–15  months) infiltrating lymphocytes (TILs), such as CD8 cytotoxic after diagnosis (t0)  (Table  2). All patients received pri- T cells and CD3 NK cells, and the Hsp70 and PD-L1 mary state-of-the-art RCT with a total radiation dose expression of the tumor. These biomarkers are usually of 69–72  Gy to the boost volume, > 49  Gy to the elec- assessed in formalin-fixed, paraffin-embedded (FFPE) tive volume and a cisplatinum-based simultaneous CT. tumor specimens of patients who underwent radical Directly after RCT all patients were checked radiologi- surgery [2, 3]. Due to the lack of tumor material after cally for accessing tumor response. Patients who did RCT there is an unmet medical need for prognostic not show tumor response after RCT are categorized as biomarkers which can be determined in the peripheral patients with “persistent tumors” and were excluded blood of patients. Radiotherapy (RT) as well as chemo- from the study. Patients with initial secondary tumors, therapy (CT) can result in drastic changes in immune- previous RT and neoadjuvant CT were not enrolled in related parameters, such as the composition, phenotype the HNprädBio study. Patients with missing data were and function of immunocompetent effector cells. u Th s, excluded from the study. Patients’ characteristics are rep- the abundance and/or activation status of these mark- resented in Tables 1 and 2. ers during therapy might provide valuable tools to pre- EDTA-blood (7.5  ml) was collected at five sequen - dict clinical outcome [4]. Therapy-induced modulations tial time points: before start of RCT (t0), after appli- in immune cell homeostasis are of great importance as cation of 20–30  Gy (t1), 3  months (t2), 6  months (t3) they might interfere with antitumor immune responses after RCT and at time of suspicion of recurrence or [5]. Therefore, we investigated the composition of major metastases, and/or after histological examination (t5, lymphocyte subsets in the peripheral blood of SCCHN 3–15  months after t0). A centralized analysis of blood patients treated with primary RCT before, during and at samples was performed according to a standard pro- different time points after treatment. tocol. All patients were followed-up until t3, only very few (non-recurrent and recurrent) patients had been Methods taken an additional blood sample after 12  months (t4). Study collective Data of recurrent patients within the study period were Thirty-two patients (n = 32), with histologically con- included, whereas data of non-recurrent patients at firmed squamous cell carcinoma of the oral cavity, time point t4 were not included into the study since N iu et al. Radiat Oncol (2021) 16:141 Page 3 of 12 the number was too small for statistical analysis. The Statistics follow up schedules were individually adapted to the Statistical differences between sets of data were evaluated patient`s risk factors. Generally, in the first two years by IBM SPSS Statistics software (IBM GmbH, Ehningen, after primary tumor occurrence clinical examina- Germany). For data sets following a normal distribution tions were performed every 3  months and radiologi- the student’s t-test was used, for all other data sets the cal diagnostics every 6  months. Then in the following Mann–Whitney Rank Sum Test was used. Values at dif- three years clinical examinations were repeated every ferent time points before, during and after RCT (t0, t1, 6 months and radiological diagnostics every 12 months. t2, t3, t5) of all patients were considered as dependent In case of suspicion of tumor recurrence during the samples; values at different time points of non-recurrent clinical examination radiological diagnostics were also and locoregional recurrent patients in comparison with performed. healthy controls were considered as independent sam- As a control (Ctrl), blood samples of twenty-two age- ples. Data sets were considered as statistically signifi - related healthy human volunteers (n = 22) with a median cantly different at p ≤ 0.05. age of 64  years (range: 27–80  years) were included into the study. This trial was approved by the ethics commit - Results − + tees of all eight DKTK partner sites and all patients and Kinetics of the proportion of CD3 /CD19 B cells before, healthy donors gave written informed consent. during RCT and in the follow‑up period Flow cytometry As summarized in Fig. 1a and Additional file  1: Table S2, − + The following major lymphocyte subpopulations were the percentage of C D3 /CD19 B cells in patients with analyzed using fluorescence-labeled antibodies (Addi - non-recurring SCCHN (n = 23) was significantly lower tional file  1: Table S1) by multiparameter flow cytometry compared to that of healthy volunteers (Ctrl) already on a FACSCalibur flow cytometer (BD Biosciences): before start of RCT at t0 (t0 vs. Ctrl: 8.88% vs. 10.83%, Leukocytes (CD45). p ≤ 0.05), and remained low until 3 months after RCT at − + B lymphocytes (CD3 /CD19 B cells). t2. The most striking drop in B cells was observed directly + − + + T lymphocytes (CD3 /CD56 T cells, CD3 /CD4 T after the application of 20–30 Gy at t1 (t0 vs. t1: 8.88% vs. + + helper cells, CD3 /CD8 cytotoxic T cells). 4.66%, p ≤ 0.001; Additional file  1: Table  S3). A recovery + + + T regulatory cells (CD4 /CD25 /FoxP3 Tregs). to initial B cell levels was detected 6  months after RCT + + Natural killer-like T (NKT) cells (C D3 /CD56 NKT at t3 (Fig.  1a, Additional file  1: Table  S2). Patients with + + cells, activated CD56 /CD69 NKT cells). locoregional recurrence did not reach initial levels up to + + Natural killer (NK) cells (activated C D56 /CD69 NK t5 (3–15  months) in case of tumor recurrence (Fig.  1a, − + − + cells, CD3 /CD56 NK cells, C D3 /CD94 NK cells, Additional file 1: Table S5). − + − + + + + CD3 /NKG2D NK cells, CD3 /NKp30 NK cells, Kinetics of the proportions of  CD3 /CD4 T helper, CD3 / − + bright/dim + + + + CD3 /NKp46 NK cells) and NK subsets (C D56 / CD8 cytotoxic and  CD4 /CD25 /FoxP3 regulatory T cell CD16 NK cells). subsets before, during RCT and in the follow‑up period + − Briefly, 100 µl of EDTA-blood was mixed with different The percentage of CD3 /CD56 T cells in SCCHN combinations of fluorescence-labeled, undiluted antibod - patients without locoregional recurrence dropped sig- ies according to the manufacturer`s recommendations. nificantly 3 (t2) and 6 (t3) months after RCT compared After incubation in the dark for 15 min, washing in phos- to initial levels (t0 vs. t2: 70.37% vs. 60.88%, p ≤ 0.01; t0 phate buffered saline (PBS)/10% heat-inactivated fetal vs. t3: 70.37% vs. 56.05%, p ≤ 0.001; Fig.  1b). At t2 and calf serum (Sigma F7524) and erythrocyte lysis buffer t3, the values were also significantly lower than those of (BD, 349,202), a total of 100.000 C D45 cells were gated controls (Ctrl vs. t2: 67.29% vs. 60.88%, p ≤ 0.05, Ctrl vs. and analyzed. In case that CD45 was not in the antibody t3: 67.29% vs. 56.05%, p ≤ 0.001; Fig. 1b, Additional file  1: panel, the data were determined on the basis of at least Table S2) and to initial levels (Additional file  1: Table S3). one additional marker that is in common with a panel In patients with locoregional recurrence T cell ratios that contains the leukocyte marker CD45. To determine showed a slight increase at t1 that dropped below initial + + the proportion of regulatory T cells, the C D4 /CD25 levels until t5 (Fig. 1b, Additional file 1: Table S4). + + gated T cell population was stained with FoxP3 anti-As CD3 /CD4 T helper cells make up more than 2/3 body after fixation (BD51-9005451) and permeabiliza - of the total CD3 T cell counts, the kinetics of these tion (BD51-9005450). The data were analyzed by using cells followed that of the T cells in the course of therapy the software BD CellQuest Pro. The ratio of positively and in the follow-up period of SCCHN patients with- stained cells is defined as the percentage of cells within out locoregional recurrence. T helper cells dropped sig- a defined lymphocyte gate minus the percentage of cells nificantly at t2 and t3 (t0 vs. t2: 46.7% vs. 25.2%, t0 vs. stained with a fluorescence labeled control antibody. t3: 46.7% vs. 26.27%, p ≤ 0.001; Fig . 1c, Additional file  1: Niu et al. Radiat Oncol (2021) 16:141 Page 4 of 12 − + + + + 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) Table S3), and patients without locoregional recurrence of cytotoxic C D8 T cells at all time points (t0, t1, had significantly lower percentages of T helper cells t2, t3) compared to controls (Ctrl vs. t0: 10.83% vs. compared to healthy controls between t1 and t3 (Ctrl 17.12%, Ctrl vs. t1: 10.83% vs. 19.03%, Ctrl vs. t2; vs. t1: 48.82% vs. 46.7%, p ≤ 0.05; Ctrl vs. t2: 48.82% vs. 10.83% vs. 25.44%, Ctrl vs. t3: 10.83% vs. 10.54%, 25.2%, Ctrl vs. t3: 48.82% vs. 26.27%, p ≤ 0.001; Fig .  1c, p ≤ 0.001; Fig .  1d, Additional file  1: Table  S2). After Additional file  1: Table  S2). Interestingly, the propor- RCT, the proportion of CD8 T cells in patients with- tion of T helper cells in patients with locoregional out locoregional recurrence increased further (t0 vs. recurrence was below that of controls and non-recur- t2: 17.12% vs. 25.44%, p ≤ 0.001; t0 vs. t3: 17.12% vs. rent patients already before start of RCT and remained 10.54%, p ≤ 0.05; Fig .  1d, Additional file  1: Table  S3). unaltered low throughout the whole course of therapy Notably, recurrent patients showed a trend towards (Fig. 1c, Additional file 1 : Tables S4, S5). higher percentages of cytotoxic T cells at all time In contrast to the T helper cells, non-recurrent SCCHN patients had significantly higher proportions N iu et al. Radiat Oncol (2021) 16:141 Page 5 of 12 Fig. 1 continued + + points (Fig. 1d, Additional file 1: Tables S4 and S5), but Kinetics of the proportion of  CD3 /CD56 NKT cells before, no significant increase after RCT. during RCT and in the follow‑up period The percentage of regulatory T cells in SCCHN Similar to CD8 T cells, NK-like T (NKT) cells increased patients without locoregional recurrence at t0 was significantly during therapy in patients without locore - lower than that in healthy controls and dropped fur- gional recurrences (t0 vs. t1: 4.41% vs. 7.18%, p ≤ 0.001; t0 ther directly after application of 20–30  Gy (t1) (Ctrl vs. t3: 4.41% vs. 7.07%, p ≤ 0.01; Fig.  1f, Additional file  1: vs. t0: 9.92% vs. 6.77%, p ≤ 0.01; Ctrl vs. t1: 9.92% vs. Table S3). Notably, before start of therapy at t0, the per- 5.98%, p ≤ 0.001; Fig .  1e, Additional file  1: Table  S2). centage of NKT cells was almost twice as high compared However, in the follow-up period (t2, t3) the percent- to healthy controls (Ctrl vs. t0: 2.46% vs. 4.41%, p ≤ 0.05, age of Tregs increased significantly above initial levels Ctrl vs. t1: 2.46% vs. 7.18%, p ≤ 0.001; Ctrl vs. t2: 2.46% (t0 vs. t2: 6.77% vs. 10.34%, p ≤ 0.01; t0 vs. t3: 6.77% vs. vs. 6.69%, p ≤ 0.001; Ctrl vs. t3: 2.46% vs. 7.07%, p ≤ 0.01; 10.06%, p ≤ 0.05; Fig . 1e, Additional file 1: Table S3). In Fig.  1f, Additional file  1: Table  S2). Patients with locore- patients with locoregional recurrence Tregs remained gional recurrences showed stably higher proportions of below that of controls throughout the course of ther- apy (Fig. 1e, Additional file 1: Table S4). Niu et al. Radiat Oncol (2021) 16:141 Page 6 of 12 Table 1 Patients’ characteristics not in patients with locoregional recurrence (Fig.  2b). Before RCT at t0 none of the tested NK cell subpopu- Total number of patients n (%) lations differed between healthy controls and patients 32 (100%) without locoregional recurrence (Fig.  2, Additional Men 27 (84.4%) file  1: Table  S6). However, the percentage of C D3 / Women 5 (15.6%) CD56 NK cells in patients without locoregional recur- Age (years) 59 (41–74) rence increased significantly after RCT (t0 vs. t2: 11.1% Tumor site vs. 15.83%, t0 vs. t3: 11.1% vs. 15.79%, p ≤ 0.01; Fig .  2b, Oral cavity 11 (34.4%) Additional file  1: Table  S7) In contrast, patients with Oropharnyx 13 (40.6%) recurrence had lower proportions of all NK cell sub- Hypopharynx 8 (25%) populations already before RCT than controls that cTNM remained at low levels at all other time points (Fig. 2b– cT2 4 (12.5%) f, Additional file 1 : Tables S8, S9). cT3 10 (31.25%) Overall, the percentages of all NK cell subsets cT4 18 (56.25%) increased significantly after RCT in patients without cN0 2 (6.25%) locoregional recurrences (Fig.  2a–f, Additional file  1: cN1 5 (15.6%) Table  S7). At t3, values of all NK cell subsets includ- cN2b 11 (34.4%) ing those bearing activating receptors such as NKG2D, cN2c 14 (43.75%) NKp30 and NKp46 were significantly higher than ini - cM0 32 (100%) tial levels and those of healthy controls (Fig.  2a–f, G2 17 (53.1%) Additional file  1: Tables S6, S7). In contrast, all NK cell G3 12 (37.5%) subgroups in recurrent patients remained below that Gx 3 (9.4%) of patients without locoregional recurrence (Fig.  2a–f, Nicotine consumption (packyears) 30.9 Additional file  1: Table  S8). Furthermore, recurrent patients had lower NK cell counts at time of locore- gional recurrence (t5) compared to patients without NKT cells than controls throughout the therapy (Fig.  1f, recurrence at t3 (Fig. 2a–f, Additional file 1 : Table S8). bright + Additional file 1: Table S4). A subgroup analysis of CD56 /CD16 NK cells in SCCHN patients without locoregional recur- Kinetics of the proportion of NK cell subsets (CD56 + / rence revealed significantly higher proportions of − + − + − + − bright + CD69 + , CD3 /CD56, CD3 /CD94, CD3 /NKG2D, CD3 / CD56 /CD16 NK cells compared to healthy con- + − + bright/dim NKp30, CD3 /NKp46) and  CD56 /CD16 NK subset trols, which dropped after application of 20–30  Gy at analysis before, during RCT and in the follow‑up period t1, but increased until t3 (Table  3). A similar course bright Already during RCT the proportion of NK cells con- was observed with respect to the CD56 /CD16-NK tinuously increased up to t3 in patients without, but cell subset in patients without locoregional recurrence. Table 2 Characteristics of recurrent SCCHN patients Recurrent patient no. Age Sex Tumor site TNM classification Time of locoregional recurrence 1 56 Female Oropharynx cT4 cN2c cM0 G2 11 2 61 Male Hypopharynx cT3 cN2c cM0 G3 6 3 61 Male Oropharynx cT4 cN2c cM0 G2 3 4 63 Female Oral cavity cT4 cN2c cM0 G2 11 5 71 Male Hypopharynx cT3 cN2b cM0 G2 8 6 52 Male Oropharynx cT4 cN2c cM0 G3 9 7 67 Male Oral cavity cT3 cN2 cM0 13 G2 8 44 Male Hypopharynx cT3 cN2b cM0 G2 15 9 73 Male Oropharynx cT3 cN0 cM0 11 G2 nr. number N iu et al. Radiat Oncol (2021) 16:141 Page 7 of 12 + + − + − + 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) bright In recurrent patients both CD56 NK cell sub- radiation since they rapidly undergo radiation-induced sets remained below that of patients without recur- apoptosis. However, the drop in B cells was not asso- rence throughout the whole course of therapy (t0–t5) ciated with a decline in the immunoglobulin levels [9], dim (Table  4). In contrast, values of the C D56 NK cell which might provide a hint that B cell activity is not subset appeared to be elevated in recurrent patients impaired by radiotherapy. Our data are in line with (Table 4). these previous findings showing a significant drop in B cells after application of 20–30  Gy during RCT Discussion and a recovery to initial levels within a time period of It has been reported that the amount of B cells that 6  months. The recovery might be mediated by B cell contribute to the humoral immune response are lower precursor cells originating from non-irradiated bone in SCCHN patients than in healthy individuals [6–8]. marrow [10] or by a retranslocation of B cells into Belka et  al. were among the first to describe B cells as the periphery. However, further studies are needed to the most sensitive lymphocyte subpopulation towards address the question whether B cell functions such as Niu et al. Radiat Oncol (2021) 16:141 Page 8 of 12 Fig. 2 continued Table 3 Significantly different values in NK cell subsets in controls (Ctrl) and non‑recurrent SCCHN patients % Ctrl t0 t1 t2 t3 bright CD56 /CD16‑ NK cells 1.83 ± 1.14 2.48 ± 1.94 1.78 ± 1.65* 2.11 ± 1.55 2.25 ± 1.62 bright + CD56 /CD16 NK cells 2.52 ± 1.34 4.72 ± 3.98* 3.72 ± 3.32 3.08 ± 2.15 4.09 ± 2.68 dim − CD56 /CD16 NK cells 2.8 ± 1.8 3.98 ± 2.82 4.42 ± 3.53 2.32 ± 1.33 7.5 ± 8.53 dim + CD56 /CD16 87.27 ± 5.97 83.10 ± 10.29 84.93 ± 10.49 89.3 ± 5.46* 82.8 ± 12.27 NK cells − + CD56 /CD16 0 ± 0 0.01 ± 0.02 0.14 ± 0.27 0.05 ± 0.09 0.09 ± 0.16 NK cells The significance is between the two underlined values. E.g. significance in CD56bright/CD16- NK cells between t0 and t1 is *p<=0.05 Composition of NK cell subsets (CD56/CD16) in % (mean value ± standard deviation) in healthy controls (Ctrl, n = 22) and non-recurrent patients (n = 23) before (t0), after application of 20–30 Gy (t1), 3 months (t2) and 6 months (t3) after RCT. Significantly different values (t0 vs. t1; Ctrl vs. t0; t0 vs. t2) are indicated in bold with an asterisk (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) antibody production or antigen presenting capacity in A long-lasting T cell imbalance with diminished secondary lymphoid organs are impaired in SCCHN T cell counts can be caused by immunosuppressive patients following RCT. effects of the tumor and its microenvironment or by N iu et al. Radiat Oncol (2021) 16:141 Page 9 of 12 Table 4 Significantly different values in NK cell subsets in controls (Ctrl) and recurrent SCCHN patients % Ctrl t0 t1 t2 t5 bright − CD56 /CD16 1.83 ± 1.14 0.92 ± 0.7* 0.75 ± 0.63* 2.07 ± 1.52 1.6 ± 1.52 NK cells bright + CD56 /CD16 2.52 ± 1.34 2.88 ± 1.85 1.47 ± 0.7* 5.76 ± 4.85 1.54 ± 1.32 NK cells dim − CD56 /CD16 2.8 ± 1.8 10.19 ± 11.85 6.08 ± 5.3 4.3 ± 3.97 2.25 ± 2.05 NK cells dim + CD56 /CD16 87.27 ± 5.97 81.4 ± 13.9 88.23 ± 7.52 84.78 ± 11.56 92.88 ± 4.26* NK cells − + CD56 /CD16 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 NK cells The significance is between the two underlined values. E.g. significance in CD56bright/CD16- NK cells between t0 and t1 is *p<=0.05 Composition of NK cell subsets (CD56/CD16) in % (mean value ± standard deviation) in healthy controls (Ctrl, n = 22) and recurrent patients (n = 9) before (t0), after application of 20–30 Gy (t1), 3 months (t2) after RCT and at time of locoregional recurrence (t5, 3–15 months after t0). Significantly different values (Ctrl vs. t0, t1; t0 vs. t1; Ctrl vs. t5) are indicated in bold with an asterisk (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) treatment-related effects [11]. Abnormalities in the T cell of an antitumor immune response induced by RCT. repertoire have been described for patients with mye- Wolf et  al. also demonstrated a weak increase in CD8 loma, breast-, ovarian- and liver cancer [12–15]. Similar T cells in head and neck cancer patients during and after to our data, Nollert et al. demonstrated a significant drop definite and adjuvant RT [15]. Moreover, Balermpas et al. + + in total C D3 T cell counts in stage IV SCCHN patients reported that elevated numbers of C D8 tumor infiltrat - by RT combined with a platinum-based chemotherapy ing lymphocytes (TILs) in SCCHN patients treated with [16]. Since cisplatinum is known to cause a systemic adjuvant RCT serve as a prognostic marker for improved toxicity which is associated with a reduced T cell prolif- clinical outcome [2]. Compared to controls, SCCHN eration, the drug (prescribed cumulative total dose of cis- patients showed significantly increased activation status 2 + + platinum was not less than 180 mg/m body surface area) of CD4 and CD8 T cells, as well as a higher migratory might be responsible for the steady decrease in CD3 T potential of these lymphocyte populations [24], which cells during and after RCT in our cohort [17, 18]. might explain an increased infiltration into the tumor tis - + + Other studies have reported that the CD4 T cell sue. However, an expansion of CD8 effector cells was counts in SCCHN patients, especially in advanced UICC accompanied by a rapid demise through apoptosis [25, stages III and IV, are significantly lower than in healthy 26]. In line with our findings, Johnson et al. observed that subjects [19, 20]. Melioli et al. observed a severe prolifer- patients with recurrent SCCHN tended to have higher + + ative defect especially in the C D4 T helper subset [21]. proportions of circulating CD8 cytotoxic T cells at diag- As T helper cells are the dominant T cell subset (up to nosis than patients who remained disease-free [23]. In 2/3 of the total T cell counts) and cisplatin’s systemic tox- addition, increased C D8 T cells in patients at diagnosis icity results in a reduced T cell proliferation [17, 18], the directly correlated with the level of tumor cell differen - + + drop in CD3 T cells is reflected in the CD4 T helper tiation and histological grading [8]. However, we could subpopulation upon RCT. In line with our findings, oth - demonstrate that the proportion of CD8 T cells did ers also observed a decrease in the T helper subset in not increase significantly upon therapy in patients with head and neck cancer patients during and after definite locoregional recurrence. and adjuvant RT [11, 15, 22]. As patients with recurrent While a high infiltration of FoxP3 Tregs at the tumor SCCHN are more likely to display abnormalities in their site correlates with an impaired overall survival in T cell development [23], the drop in CD4 T cells before patients with melanomas, cervical-, renal- and breast start of therapy (t0) appeared to be more pronounced cancers an opposite effect was observed in colorectal, in patients with locoregional recurrence compared to head and neck as well as esophageal cancers [27]. As head patients who remained free of recurrence [22–24]. There - and neck cancer, colon cancer and hematologic malig- fore, we hypothesize that low initial T helper counts nancies are among those tumors which are heavily infil - might provide a potential predictive marker for patients trated by immune cells that facilitate tumor progression that more likely will develop a recurrent disease. by producing growth factors and/or proinflammatory A significant increase in the percentage of CD8 T cytokines, Tregs might limit tumor-promoting inflam - cells in SCCHN patients without locoregional recur- mation by the release of immunosuppressive cytokines rence upon RCT might be indicative for the induction [28–31]. Similar to our results, Lee et  al. observed a Niu et al. Radiat Oncol (2021) 16:141 Page 10 of 12 + + + values remained elevated in patients without locore- weak decline in CD4 /CD25 /FoxP3 Tregs in patients gional recurrence throughout the course of therapy and with oral squamous cell carcinoma compared to healthy in the follow-up period. Activation of NK/NKT cells is controls [24], while a rise in the frequency of Tregs was associated with an increase in the early activation marker found in patients with SCCHN treated with adjuvant CD69 in patients without locoregional recurrence. A RCT [5, 32]. Finally, Kachikwu et al. observed an increase high NK cell activity has been shown to correlate with in Tregs after radiation which was associated with a more lower incidence of tumors and thereby improves, along- radioresistant phenotype [33]. side with NK cell infiltration in SCCHN, patients’ clini - Kobayashi et al. described the ability of radiotherapy to cal outcome [41, 42]. Similar to our findings, Takeuchhi improve proliferation and antitumor immunity of NKT et  al. demonstrated that a low NK cell activity leads to cells [34]. In our SCCHN cohort, especially patients with a higher incidence of tumor occurrence and metastasis, locoregional recurrences, exhibited increased NKT cell and its degree correlates with an increased invasiveness levels at diagnosis which further increase throughout of the tumor [43]. In line with these findings, impairment therapy as compared to controls. These data suggest the of NK cell function is greater in patients with advanced presence of a predefined immune imbalance which cor - tumors, as primary squamous cell carcinoma patients in relates with treatment response and the ability of RCT stages T3-T4 showed lower NK cell activity than patients to boost SCCHN patients’ immunocompetence. Addi- in earlier stages T1-T2 [44, 45]. Ye et  al. observed a sig- tionally, the particularly high NKT cell value in recurrent + + nificant decrease in CD56 /CD16 circulating NK cells patients at t0 could provide a hint for an increased risk in oral cancer patients after surgery [46], indicating that for an early recurrence. not only RCT, but also inflammatory effects induced by In our study, a significant increase in the percentage surgery may have an impact on the NK cell hemostasis. of NK cells with activating receptors such as NKG2D, In our study, the proportion of all activated NK cell sub- NKp30 and NKp46 might reflect the effect of RCT on sets was lower in the recurrent patient group compared patients’ immunocompetence to improve antitumor to healthy individuals and patients without locoregional responsiveness. Moreover, it also has been reported that recurrence. Activatory NK cell subsets increased signifi - cytotoxic lymphocytes, including CD8 T cells and NK cantly in the course of therapy in non-recurrent patients, cells, contribute to the efficacy of certain chemothera - but remained nearly unaltered until t5 in patients with peutic drugs [4], such as cisplatinum, paclitaxel and locoregional recurrence. Since NK cell responses occur doxorubicin, which have been shown to enhance the fast this finding might be of relevance for predicting a NK and C D8 T cell-mediated killing as they increase potential recurrence and to adjust the treatment at an the permeability of tumor cells towards the apoptosis earlier time point. Our findings demonstrated the ability inducing serine protease granzyme B [35]. Recurrent of RCT in enhancing antitumor immune responses medi- patients, in particular at diagnosis, tended to have much ated by NK cells in patients with SCCHN. Therefore, we lower NK cell counts than healthy controls and patients speculate that a combination of RCT with immunother- without recurrence at all time points. An impaired first apy such as immune checkpoint inhibitors might provide line defense might indicate an imbalance of the innate a promising strategy to increase antitumor activity of NK immune cell function in patients with recurrence. bright + cells in patients with SCCHN after RCT who are on high CD56 /CD16 NK cells are considered to have a risk to develop recurrences. low cytotoxic activity and a high cytokine/chemokine dim + production capacity, whereas the CD56 /CD16 NK cell population is associated with a high cytotoxic Conclusions potential [36–38]. In accordance with this hypothesis, We could show altered lymphocyte compositions in Mamessier et  al. observed an increased proportion of bright + patients with non-locoregional recurrences and patients CD56 /CD16 circulating NK cells in advanced with locoregional recurrences after primary RCT. These breast cancer patients and patients with metastases changes already existed before start of therapy and per- [39]. However, more recent data by the group of Wagner bright + sisted for months in the follow-up period. B cells, T et  al. revealed that upon stimulation C D56 /CD16 cells, T helper cells and regulatory T cells dropped sig- NK cells can also exert direct antitumor activity [40]. bright + nificantly after RCT, but unlike T and T helper cells, B CD56 /CD16 NK cells mediate strong cytotoxic- cells and regulatory T cells recovered to initial levels ity against MHC class I negative as well as MHC class I 6  months after RCT. Inversely, cytotoxic T cells, NKT positive tumors [38]. Our findings indicate significant bright + and especially activating C D3 NK cell subsets gradu- higher percentages of C D56 /CD16 NK cells in ally increased in the same time period in non-recurrent patients without locoregional recurrence compared to patients, but remained nearly unaltered in patients healthy controls and patients with recurrence. These N iu et al. Radiat Oncol (2021) 16:141 Page 11 of 12 OncoRay – National Centre for Radiation Research in Oncology, Faculty with recurrent tumors. Following validation of our data of Medicine and University Hospital Carl Gustav Carus, Technische Universität in a larger patient cohort, monitoring cellular immu- Dresden, Helmholtz‑Zentrum Dresden, Rossendorf, Dresden, Germany. Ger‑ nity in the peripheral blood of SCCHN patients during man Cancer Consortium (DKTK), Partner Site Dresden, Germany. Depar t‑ ment of Radiotherapy and Radiation Oncology, Faculty of Medicine, University therapy might offer an opportunity for an early evalu - Hospital Carl Gustav Carus, Technische Universität, Dresden, Germany. ation of treatment outcome and might help to stratify National Center for Tumor Diseases (NCT ), Partner Site Dresden, Germany. for patients that most likely will benefit from an additive Faculty of Medicine and University Hospital, Partner Site Dresden, Germany. Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. immunotherapy. Helmholtz Association/Helmholtz‑Zentrum Dresden – Rossendorf (HZDR), 17 18 Dresden, Germany. OncoRay, Dresden, Germany. Department of Radiation Oncology, Medical Centre University of Freiburg, Freiburg, Germany. German Abbreviations Cancer Consortium (DKTK), Partner Site Freiburg, Germany. SCCHN: Squamous cell carcinoma of the head and neck; RCT : Radiochemo‑ therapy; CT: Chemotherapy; RT: Radiotherapy; NK: Natural killer cells; NKT: Received: 21 December 2020 Accepted: 19 July 2021 Natural killer‑like T cells; MHC: Major histocompatibility complex. Supplementary Information The online version contains supplementary material available at https:// doi. References org/ 10. 1186/ s13014‑ 021‑ 01868‑5. 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major Additional file 1. Supplementary materials. patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359‑386. 2. Balermpas P, Rödel F, Rödel C, Krause M, Linge A, Lohaus F, et al. 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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)

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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 volun‑ teers (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 *Correspondence: minli.niu@tum.de Center for Translational Cancer Research ( TranslaTUM), Radiation Immuno‑Oncology Group, Klinikum rechts der isar, TU München ( TUM), Einsteinstr. 25, 81675 Munich, Germany Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Niu et al. Radiat Oncol (2021) 16:141 Page 2 of 12 + + + 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:// clini caltr ials. gov/ ct2/ show/ NCT02 059668. Keywords: SCCHN, Prediction of locoregional recurrence, Immunophenotyping, Radiochemotherapy, Lymphocyte subpopulations, NK cell subsets Background oro- and hypopharynx at UICC stadium III or IV with- Head and neck cancer is the seventh most common can- out any remote metastases were included into the cer worldwide with an incidence rate of approximately study  (Table  1). The patients are a subset of patients 600.000 new cases in 2012 [1]. Despite progress in radia- recruited into the HNprädBio study (clinicaltrials. tion oncology over the past decades, the 5  year survival gov, NCT02059668) of the German Cancer Consor- rate of 40–60% of patients with locally advanced SCCHN tium Radiation Oncology Group (DKTK-ROG), which is still not satisfying [1]. In advanced inoperable stages, is based on the availability of blood samples in the time SCCHN patients are treated primarily with RCT. Poten- period from May 2014 till December 2016. Nine of these tial prognostic markers for the response to RCT include patients developed a locoregional recurrence within a the human papillomavirus (HPV) infection status, tumor median time period of 11  months (range: 3–15  months) infiltrating lymphocytes (TILs), such as CD8 cytotoxic after diagnosis (t0)  (Table  2). All patients received pri- T cells and CD3 NK cells, and the Hsp70 and PD-L1 mary state-of-the-art RCT with a total radiation dose expression of the tumor. These biomarkers are usually of 69–72  Gy to the boost volume, > 49  Gy to the elec- assessed in formalin-fixed, paraffin-embedded (FFPE) tive volume and a cisplatinum-based simultaneous CT. tumor specimens of patients who underwent radical Directly after RCT all patients were checked radiologi- surgery [2, 3]. Due to the lack of tumor material after cally for accessing tumor response. Patients who did RCT there is an unmet medical need for prognostic not show tumor response after RCT are categorized as biomarkers which can be determined in the peripheral patients with “persistent tumors” and were excluded blood of patients. Radiotherapy (RT) as well as chemo- from the study. Patients with initial secondary tumors, therapy (CT) can result in drastic changes in immune- previous RT and neoadjuvant CT were not enrolled in related parameters, such as the composition, phenotype the HNprädBio study. Patients with missing data were and function of immunocompetent effector cells. u Th s, excluded from the study. Patients’ characteristics are rep- the abundance and/or activation status of these mark- resented in Tables 1 and 2. ers during therapy might provide valuable tools to pre- EDTA-blood (7.5  ml) was collected at five sequen - dict clinical outcome [4]. Therapy-induced modulations tial time points: before start of RCT (t0), after appli- in immune cell homeostasis are of great importance as cation of 20–30  Gy (t1), 3  months (t2), 6  months (t3) they might interfere with antitumor immune responses after RCT and at time of suspicion of recurrence or [5]. Therefore, we investigated the composition of major metastases, and/or after histological examination (t5, lymphocyte subsets in the peripheral blood of SCCHN 3–15  months after t0). A centralized analysis of blood patients treated with primary RCT before, during and at samples was performed according to a standard pro- different time points after treatment. tocol. All patients were followed-up until t3, only very few (non-recurrent and recurrent) patients had been Methods taken an additional blood sample after 12  months (t4). Study collective Data of recurrent patients within the study period were Thirty-two patients (n = 32), with histologically con- included, whereas data of non-recurrent patients at firmed squamous cell carcinoma of the oral cavity, time point t4 were not included into the study since N iu et al. Radiat Oncol (2021) 16:141 Page 3 of 12 the number was too small for statistical analysis. The Statistics follow up schedules were individually adapted to the Statistical differences between sets of data were evaluated patient`s risk factors. Generally, in the first two years by IBM SPSS Statistics software (IBM GmbH, Ehningen, after primary tumor occurrence clinical examina- Germany). For data sets following a normal distribution tions were performed every 3  months and radiologi- the student’s t-test was used, for all other data sets the cal diagnostics every 6  months. Then in the following Mann–Whitney Rank Sum Test was used. Values at dif- three years clinical examinations were repeated every ferent time points before, during and after RCT (t0, t1, 6 months and radiological diagnostics every 12 months. t2, t3, t5) of all patients were considered as dependent In case of suspicion of tumor recurrence during the samples; values at different time points of non-recurrent clinical examination radiological diagnostics were also and locoregional recurrent patients in comparison with performed. healthy controls were considered as independent sam- As a control (Ctrl), blood samples of twenty-two age- ples. Data sets were considered as statistically signifi - related healthy human volunteers (n = 22) with a median cantly different at p ≤ 0.05. age of 64  years (range: 27–80  years) were included into the study. This trial was approved by the ethics commit - Results − + tees of all eight DKTK partner sites and all patients and Kinetics of the proportion of CD3 /CD19 B cells before, healthy donors gave written informed consent. during RCT and in the follow‑up period Flow cytometry As summarized in Fig. 1a and Additional file  1: Table S2, − + The following major lymphocyte subpopulations were the percentage of C D3 /CD19 B cells in patients with analyzed using fluorescence-labeled antibodies (Addi - non-recurring SCCHN (n = 23) was significantly lower tional file  1: Table S1) by multiparameter flow cytometry compared to that of healthy volunteers (Ctrl) already on a FACSCalibur flow cytometer (BD Biosciences): before start of RCT at t0 (t0 vs. Ctrl: 8.88% vs. 10.83%, Leukocytes (CD45). p ≤ 0.05), and remained low until 3 months after RCT at − + B lymphocytes (CD3 /CD19 B cells). t2. The most striking drop in B cells was observed directly + − + + T lymphocytes (CD3 /CD56 T cells, CD3 /CD4 T after the application of 20–30 Gy at t1 (t0 vs. t1: 8.88% vs. + + helper cells, CD3 /CD8 cytotoxic T cells). 4.66%, p ≤ 0.001; Additional file  1: Table  S3). A recovery + + + T regulatory cells (CD4 /CD25 /FoxP3 Tregs). to initial B cell levels was detected 6  months after RCT + + Natural killer-like T (NKT) cells (C D3 /CD56 NKT at t3 (Fig.  1a, Additional file  1: Table  S2). Patients with + + cells, activated CD56 /CD69 NKT cells). locoregional recurrence did not reach initial levels up to + + Natural killer (NK) cells (activated C D56 /CD69 NK t5 (3–15  months) in case of tumor recurrence (Fig.  1a, − + − + cells, CD3 /CD56 NK cells, C D3 /CD94 NK cells, Additional file 1: Table S5). − + − + + + + CD3 /NKG2D NK cells, CD3 /NKp30 NK cells, Kinetics of the proportions of  CD3 /CD4 T helper, CD3 / − + bright/dim + + + + CD3 /NKp46 NK cells) and NK subsets (C D56 / CD8 cytotoxic and  CD4 /CD25 /FoxP3 regulatory T cell CD16 NK cells). subsets before, during RCT and in the follow‑up period + − Briefly, 100 µl of EDTA-blood was mixed with different The percentage of CD3 /CD56 T cells in SCCHN combinations of fluorescence-labeled, undiluted antibod - patients without locoregional recurrence dropped sig- ies according to the manufacturer`s recommendations. nificantly 3 (t2) and 6 (t3) months after RCT compared After incubation in the dark for 15 min, washing in phos- to initial levels (t0 vs. t2: 70.37% vs. 60.88%, p ≤ 0.01; t0 phate buffered saline (PBS)/10% heat-inactivated fetal vs. t3: 70.37% vs. 56.05%, p ≤ 0.001; Fig.  1b). At t2 and calf serum (Sigma F7524) and erythrocyte lysis buffer t3, the values were also significantly lower than those of (BD, 349,202), a total of 100.000 C D45 cells were gated controls (Ctrl vs. t2: 67.29% vs. 60.88%, p ≤ 0.05, Ctrl vs. and analyzed. In case that CD45 was not in the antibody t3: 67.29% vs. 56.05%, p ≤ 0.001; Fig. 1b, Additional file  1: panel, the data were determined on the basis of at least Table S2) and to initial levels (Additional file  1: Table S3). one additional marker that is in common with a panel In patients with locoregional recurrence T cell ratios that contains the leukocyte marker CD45. To determine showed a slight increase at t1 that dropped below initial + + the proportion of regulatory T cells, the C D4 /CD25 levels until t5 (Fig. 1b, Additional file 1: Table S4). + + gated T cell population was stained with FoxP3 anti-As CD3 /CD4 T helper cells make up more than 2/3 body after fixation (BD51-9005451) and permeabiliza - of the total CD3 T cell counts, the kinetics of these tion (BD51-9005450). The data were analyzed by using cells followed that of the T cells in the course of therapy the software BD CellQuest Pro. The ratio of positively and in the follow-up period of SCCHN patients with- stained cells is defined as the percentage of cells within out locoregional recurrence. T helper cells dropped sig- a defined lymphocyte gate minus the percentage of cells nificantly at t2 and t3 (t0 vs. t2: 46.7% vs. 25.2%, t0 vs. stained with a fluorescence labeled control antibody. t3: 46.7% vs. 26.27%, p ≤ 0.001; Fig . 1c, Additional file  1: Niu et al. Radiat Oncol (2021) 16:141 Page 4 of 12 − + + + + 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) Table S3), and patients without locoregional recurrence of cytotoxic C D8 T cells at all time points (t0, t1, had significantly lower percentages of T helper cells t2, t3) compared to controls (Ctrl vs. t0: 10.83% vs. compared to healthy controls between t1 and t3 (Ctrl 17.12%, Ctrl vs. t1: 10.83% vs. 19.03%, Ctrl vs. t2; vs. t1: 48.82% vs. 46.7%, p ≤ 0.05; Ctrl vs. t2: 48.82% vs. 10.83% vs. 25.44%, Ctrl vs. t3: 10.83% vs. 10.54%, 25.2%, Ctrl vs. t3: 48.82% vs. 26.27%, p ≤ 0.001; Fig .  1c, p ≤ 0.001; Fig .  1d, Additional file  1: Table  S2). After Additional file  1: Table  S2). Interestingly, the propor- RCT, the proportion of CD8 T cells in patients with- tion of T helper cells in patients with locoregional out locoregional recurrence increased further (t0 vs. recurrence was below that of controls and non-recur- t2: 17.12% vs. 25.44%, p ≤ 0.001; t0 vs. t3: 17.12% vs. rent patients already before start of RCT and remained 10.54%, p ≤ 0.05; Fig .  1d, Additional file  1: Table  S3). unaltered low throughout the whole course of therapy Notably, recurrent patients showed a trend towards (Fig. 1c, Additional file 1 : Tables S4, S5). higher percentages of cytotoxic T cells at all time In contrast to the T helper cells, non-recurrent SCCHN patients had significantly higher proportions N iu et al. Radiat Oncol (2021) 16:141 Page 5 of 12 Fig. 1 continued + + points (Fig. 1d, Additional file 1: Tables S4 and S5), but Kinetics of the proportion of  CD3 /CD56 NKT cells before, no significant increase after RCT. during RCT and in the follow‑up period The percentage of regulatory T cells in SCCHN Similar to CD8 T cells, NK-like T (NKT) cells increased patients without locoregional recurrence at t0 was significantly during therapy in patients without locore - lower than that in healthy controls and dropped fur- gional recurrences (t0 vs. t1: 4.41% vs. 7.18%, p ≤ 0.001; t0 ther directly after application of 20–30  Gy (t1) (Ctrl vs. t3: 4.41% vs. 7.07%, p ≤ 0.01; Fig.  1f, Additional file  1: vs. t0: 9.92% vs. 6.77%, p ≤ 0.01; Ctrl vs. t1: 9.92% vs. Table S3). Notably, before start of therapy at t0, the per- 5.98%, p ≤ 0.001; Fig .  1e, Additional file  1: Table  S2). centage of NKT cells was almost twice as high compared However, in the follow-up period (t2, t3) the percent- to healthy controls (Ctrl vs. t0: 2.46% vs. 4.41%, p ≤ 0.05, age of Tregs increased significantly above initial levels Ctrl vs. t1: 2.46% vs. 7.18%, p ≤ 0.001; Ctrl vs. t2: 2.46% (t0 vs. t2: 6.77% vs. 10.34%, p ≤ 0.01; t0 vs. t3: 6.77% vs. vs. 6.69%, p ≤ 0.001; Ctrl vs. t3: 2.46% vs. 7.07%, p ≤ 0.01; 10.06%, p ≤ 0.05; Fig . 1e, Additional file 1: Table S3). In Fig.  1f, Additional file  1: Table  S2). Patients with locore- patients with locoregional recurrence Tregs remained gional recurrences showed stably higher proportions of below that of controls throughout the course of ther- apy (Fig. 1e, Additional file 1: Table S4). Niu et al. Radiat Oncol (2021) 16:141 Page 6 of 12 Table 1 Patients’ characteristics not in patients with locoregional recurrence (Fig.  2b). Before RCT at t0 none of the tested NK cell subpopu- Total number of patients n (%) lations differed between healthy controls and patients 32 (100%) without locoregional recurrence (Fig.  2, Additional Men 27 (84.4%) file  1: Table  S6). However, the percentage of C D3 / Women 5 (15.6%) CD56 NK cells in patients without locoregional recur- Age (years) 59 (41–74) rence increased significantly after RCT (t0 vs. t2: 11.1% Tumor site vs. 15.83%, t0 vs. t3: 11.1% vs. 15.79%, p ≤ 0.01; Fig .  2b, Oral cavity 11 (34.4%) Additional file  1: Table  S7) In contrast, patients with Oropharnyx 13 (40.6%) recurrence had lower proportions of all NK cell sub- Hypopharynx 8 (25%) populations already before RCT than controls that cTNM remained at low levels at all other time points (Fig. 2b– cT2 4 (12.5%) f, Additional file 1 : Tables S8, S9). cT3 10 (31.25%) Overall, the percentages of all NK cell subsets cT4 18 (56.25%) increased significantly after RCT in patients without cN0 2 (6.25%) locoregional recurrences (Fig.  2a–f, Additional file  1: cN1 5 (15.6%) Table  S7). At t3, values of all NK cell subsets includ- cN2b 11 (34.4%) ing those bearing activating receptors such as NKG2D, cN2c 14 (43.75%) NKp30 and NKp46 were significantly higher than ini - cM0 32 (100%) tial levels and those of healthy controls (Fig.  2a–f, G2 17 (53.1%) Additional file  1: Tables S6, S7). In contrast, all NK cell G3 12 (37.5%) subgroups in recurrent patients remained below that Gx 3 (9.4%) of patients without locoregional recurrence (Fig.  2a–f, Nicotine consumption (packyears) 30.9 Additional file  1: Table  S8). Furthermore, recurrent patients had lower NK cell counts at time of locore- gional recurrence (t5) compared to patients without NKT cells than controls throughout the therapy (Fig.  1f, recurrence at t3 (Fig. 2a–f, Additional file 1 : Table S8). bright + Additional file 1: Table S4). A subgroup analysis of CD56 /CD16 NK cells in SCCHN patients without locoregional recur- Kinetics of the proportion of NK cell subsets (CD56 + / rence revealed significantly higher proportions of − + − + − + − bright + CD69 + , CD3 /CD56, CD3 /CD94, CD3 /NKG2D, CD3 / CD56 /CD16 NK cells compared to healthy con- + − + bright/dim NKp30, CD3 /NKp46) and  CD56 /CD16 NK subset trols, which dropped after application of 20–30  Gy at analysis before, during RCT and in the follow‑up period t1, but increased until t3 (Table  3). A similar course bright Already during RCT the proportion of NK cells con- was observed with respect to the CD56 /CD16-NK tinuously increased up to t3 in patients without, but cell subset in patients without locoregional recurrence. Table 2 Characteristics of recurrent SCCHN patients Recurrent patient no. Age Sex Tumor site TNM classification Time of locoregional recurrence 1 56 Female Oropharynx cT4 cN2c cM0 G2 11 2 61 Male Hypopharynx cT3 cN2c cM0 G3 6 3 61 Male Oropharynx cT4 cN2c cM0 G2 3 4 63 Female Oral cavity cT4 cN2c cM0 G2 11 5 71 Male Hypopharynx cT3 cN2b cM0 G2 8 6 52 Male Oropharynx cT4 cN2c cM0 G3 9 7 67 Male Oral cavity cT3 cN2 cM0 13 G2 8 44 Male Hypopharynx cT3 cN2b cM0 G2 15 9 73 Male Oropharynx cT3 cN0 cM0 11 G2 nr. number N iu et al. Radiat Oncol (2021) 16:141 Page 7 of 12 + + − + − + 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) bright In recurrent patients both CD56 NK cell sub- radiation since they rapidly undergo radiation-induced sets remained below that of patients without recur- apoptosis. However, the drop in B cells was not asso- rence throughout the whole course of therapy (t0–t5) ciated with a decline in the immunoglobulin levels [9], dim (Table  4). In contrast, values of the C D56 NK cell which might provide a hint that B cell activity is not subset appeared to be elevated in recurrent patients impaired by radiotherapy. Our data are in line with (Table 4). these previous findings showing a significant drop in B cells after application of 20–30  Gy during RCT Discussion and a recovery to initial levels within a time period of It has been reported that the amount of B cells that 6  months. The recovery might be mediated by B cell contribute to the humoral immune response are lower precursor cells originating from non-irradiated bone in SCCHN patients than in healthy individuals [6–8]. marrow [10] or by a retranslocation of B cells into Belka et  al. were among the first to describe B cells as the periphery. However, further studies are needed to the most sensitive lymphocyte subpopulation towards address the question whether B cell functions such as Niu et al. Radiat Oncol (2021) 16:141 Page 8 of 12 Fig. 2 continued Table 3 Significantly different values in NK cell subsets in controls (Ctrl) and non‑recurrent SCCHN patients % Ctrl t0 t1 t2 t3 bright CD56 /CD16‑ NK cells 1.83 ± 1.14 2.48 ± 1.94 1.78 ± 1.65* 2.11 ± 1.55 2.25 ± 1.62 bright + CD56 /CD16 NK cells 2.52 ± 1.34 4.72 ± 3.98* 3.72 ± 3.32 3.08 ± 2.15 4.09 ± 2.68 dim − CD56 /CD16 NK cells 2.8 ± 1.8 3.98 ± 2.82 4.42 ± 3.53 2.32 ± 1.33 7.5 ± 8.53 dim + CD56 /CD16 87.27 ± 5.97 83.10 ± 10.29 84.93 ± 10.49 89.3 ± 5.46* 82.8 ± 12.27 NK cells − + CD56 /CD16 0 ± 0 0.01 ± 0.02 0.14 ± 0.27 0.05 ± 0.09 0.09 ± 0.16 NK cells The significance is between the two underlined values. E.g. significance in CD56bright/CD16- NK cells between t0 and t1 is *p<=0.05 Composition of NK cell subsets (CD56/CD16) in % (mean value ± standard deviation) in healthy controls (Ctrl, n = 22) and non-recurrent patients (n = 23) before (t0), after application of 20–30 Gy (t1), 3 months (t2) and 6 months (t3) after RCT. Significantly different values (t0 vs. t1; Ctrl vs. t0; t0 vs. t2) are indicated in bold with an asterisk (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) antibody production or antigen presenting capacity in A long-lasting T cell imbalance with diminished secondary lymphoid organs are impaired in SCCHN T cell counts can be caused by immunosuppressive patients following RCT. effects of the tumor and its microenvironment or by N iu et al. Radiat Oncol (2021) 16:141 Page 9 of 12 Table 4 Significantly different values in NK cell subsets in controls (Ctrl) and recurrent SCCHN patients % Ctrl t0 t1 t2 t5 bright − CD56 /CD16 1.83 ± 1.14 0.92 ± 0.7* 0.75 ± 0.63* 2.07 ± 1.52 1.6 ± 1.52 NK cells bright + CD56 /CD16 2.52 ± 1.34 2.88 ± 1.85 1.47 ± 0.7* 5.76 ± 4.85 1.54 ± 1.32 NK cells dim − CD56 /CD16 2.8 ± 1.8 10.19 ± 11.85 6.08 ± 5.3 4.3 ± 3.97 2.25 ± 2.05 NK cells dim + CD56 /CD16 87.27 ± 5.97 81.4 ± 13.9 88.23 ± 7.52 84.78 ± 11.56 92.88 ± 4.26* NK cells − + CD56 /CD16 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 NK cells The significance is between the two underlined values. E.g. significance in CD56bright/CD16- NK cells between t0 and t1 is *p<=0.05 Composition of NK cell subsets (CD56/CD16) in % (mean value ± standard deviation) in healthy controls (Ctrl, n = 22) and recurrent patients (n = 9) before (t0), after application of 20–30 Gy (t1), 3 months (t2) after RCT and at time of locoregional recurrence (t5, 3–15 months after t0). Significantly different values (Ctrl vs. t0, t1; t0 vs. t1; Ctrl vs. t5) are indicated in bold with an asterisk (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) treatment-related effects [11]. Abnormalities in the T cell of an antitumor immune response induced by RCT. repertoire have been described for patients with mye- Wolf et  al. also demonstrated a weak increase in CD8 loma, breast-, ovarian- and liver cancer [12–15]. Similar T cells in head and neck cancer patients during and after to our data, Nollert et al. demonstrated a significant drop definite and adjuvant RT [15]. Moreover, Balermpas et al. + + in total C D3 T cell counts in stage IV SCCHN patients reported that elevated numbers of C D8 tumor infiltrat - by RT combined with a platinum-based chemotherapy ing lymphocytes (TILs) in SCCHN patients treated with [16]. Since cisplatinum is known to cause a systemic adjuvant RCT serve as a prognostic marker for improved toxicity which is associated with a reduced T cell prolif- clinical outcome [2]. Compared to controls, SCCHN eration, the drug (prescribed cumulative total dose of cis- patients showed significantly increased activation status 2 + + platinum was not less than 180 mg/m body surface area) of CD4 and CD8 T cells, as well as a higher migratory might be responsible for the steady decrease in CD3 T potential of these lymphocyte populations [24], which cells during and after RCT in our cohort [17, 18]. might explain an increased infiltration into the tumor tis - + + Other studies have reported that the CD4 T cell sue. However, an expansion of CD8 effector cells was counts in SCCHN patients, especially in advanced UICC accompanied by a rapid demise through apoptosis [25, stages III and IV, are significantly lower than in healthy 26]. In line with our findings, Johnson et al. observed that subjects [19, 20]. Melioli et al. observed a severe prolifer- patients with recurrent SCCHN tended to have higher + + ative defect especially in the C D4 T helper subset [21]. proportions of circulating CD8 cytotoxic T cells at diag- As T helper cells are the dominant T cell subset (up to nosis than patients who remained disease-free [23]. In 2/3 of the total T cell counts) and cisplatin’s systemic tox- addition, increased C D8 T cells in patients at diagnosis icity results in a reduced T cell proliferation [17, 18], the directly correlated with the level of tumor cell differen - + + drop in CD3 T cells is reflected in the CD4 T helper tiation and histological grading [8]. However, we could subpopulation upon RCT. In line with our findings, oth - demonstrate that the proportion of CD8 T cells did ers also observed a decrease in the T helper subset in not increase significantly upon therapy in patients with head and neck cancer patients during and after definite locoregional recurrence. and adjuvant RT [11, 15, 22]. As patients with recurrent While a high infiltration of FoxP3 Tregs at the tumor SCCHN are more likely to display abnormalities in their site correlates with an impaired overall survival in T cell development [23], the drop in CD4 T cells before patients with melanomas, cervical-, renal- and breast start of therapy (t0) appeared to be more pronounced cancers an opposite effect was observed in colorectal, in patients with locoregional recurrence compared to head and neck as well as esophageal cancers [27]. As head patients who remained free of recurrence [22–24]. There - and neck cancer, colon cancer and hematologic malig- fore, we hypothesize that low initial T helper counts nancies are among those tumors which are heavily infil - might provide a potential predictive marker for patients trated by immune cells that facilitate tumor progression that more likely will develop a recurrent disease. by producing growth factors and/or proinflammatory A significant increase in the percentage of CD8 T cytokines, Tregs might limit tumor-promoting inflam - cells in SCCHN patients without locoregional recur- mation by the release of immunosuppressive cytokines rence upon RCT might be indicative for the induction [28–31]. Similar to our results, Lee et  al. observed a Niu et al. Radiat Oncol (2021) 16:141 Page 10 of 12 + + + values remained elevated in patients without locore- weak decline in CD4 /CD25 /FoxP3 Tregs in patients gional recurrence throughout the course of therapy and with oral squamous cell carcinoma compared to healthy in the follow-up period. Activation of NK/NKT cells is controls [24], while a rise in the frequency of Tregs was associated with an increase in the early activation marker found in patients with SCCHN treated with adjuvant CD69 in patients without locoregional recurrence. A RCT [5, 32]. Finally, Kachikwu et al. observed an increase high NK cell activity has been shown to correlate with in Tregs after radiation which was associated with a more lower incidence of tumors and thereby improves, along- radioresistant phenotype [33]. side with NK cell infiltration in SCCHN, patients’ clini - Kobayashi et al. described the ability of radiotherapy to cal outcome [41, 42]. Similar to our findings, Takeuchhi improve proliferation and antitumor immunity of NKT et  al. demonstrated that a low NK cell activity leads to cells [34]. In our SCCHN cohort, especially patients with a higher incidence of tumor occurrence and metastasis, locoregional recurrences, exhibited increased NKT cell and its degree correlates with an increased invasiveness levels at diagnosis which further increase throughout of the tumor [43]. In line with these findings, impairment therapy as compared to controls. These data suggest the of NK cell function is greater in patients with advanced presence of a predefined immune imbalance which cor - tumors, as primary squamous cell carcinoma patients in relates with treatment response and the ability of RCT stages T3-T4 showed lower NK cell activity than patients to boost SCCHN patients’ immunocompetence. Addi- in earlier stages T1-T2 [44, 45]. Ye et  al. observed a sig- tionally, the particularly high NKT cell value in recurrent + + nificant decrease in CD56 /CD16 circulating NK cells patients at t0 could provide a hint for an increased risk in oral cancer patients after surgery [46], indicating that for an early recurrence. not only RCT, but also inflammatory effects induced by In our study, a significant increase in the percentage surgery may have an impact on the NK cell hemostasis. of NK cells with activating receptors such as NKG2D, In our study, the proportion of all activated NK cell sub- NKp30 and NKp46 might reflect the effect of RCT on sets was lower in the recurrent patient group compared patients’ immunocompetence to improve antitumor to healthy individuals and patients without locoregional responsiveness. Moreover, it also has been reported that recurrence. Activatory NK cell subsets increased signifi - cytotoxic lymphocytes, including CD8 T cells and NK cantly in the course of therapy in non-recurrent patients, cells, contribute to the efficacy of certain chemothera - but remained nearly unaltered until t5 in patients with peutic drugs [4], such as cisplatinum, paclitaxel and locoregional recurrence. Since NK cell responses occur doxorubicin, which have been shown to enhance the fast this finding might be of relevance for predicting a NK and C D8 T cell-mediated killing as they increase potential recurrence and to adjust the treatment at an the permeability of tumor cells towards the apoptosis earlier time point. Our findings demonstrated the ability inducing serine protease granzyme B [35]. Recurrent of RCT in enhancing antitumor immune responses medi- patients, in particular at diagnosis, tended to have much ated by NK cells in patients with SCCHN. Therefore, we lower NK cell counts than healthy controls and patients speculate that a combination of RCT with immunother- without recurrence at all time points. An impaired first apy such as immune checkpoint inhibitors might provide line defense might indicate an imbalance of the innate a promising strategy to increase antitumor activity of NK immune cell function in patients with recurrence. bright + cells in patients with SCCHN after RCT who are on high CD56 /CD16 NK cells are considered to have a risk to develop recurrences. low cytotoxic activity and a high cytokine/chemokine dim + production capacity, whereas the CD56 /CD16 NK cell population is associated with a high cytotoxic Conclusions potential [36–38]. In accordance with this hypothesis, We could show altered lymphocyte compositions in Mamessier et  al. observed an increased proportion of bright + patients with non-locoregional recurrences and patients CD56 /CD16 circulating NK cells in advanced with locoregional recurrences after primary RCT. These breast cancer patients and patients with metastases changes already existed before start of therapy and per- [39]. However, more recent data by the group of Wagner bright + sisted for months in the follow-up period. B cells, T et  al. revealed that upon stimulation C D56 /CD16 cells, T helper cells and regulatory T cells dropped sig- NK cells can also exert direct antitumor activity [40]. bright + nificantly after RCT, but unlike T and T helper cells, B CD56 /CD16 NK cells mediate strong cytotoxic- cells and regulatory T cells recovered to initial levels ity against MHC class I negative as well as MHC class I 6  months after RCT. Inversely, cytotoxic T cells, NKT positive tumors [38]. Our findings indicate significant bright + and especially activating C D3 NK cell subsets gradu- higher percentages of C D56 /CD16 NK cells in ally increased in the same time period in non-recurrent patients without locoregional recurrence compared to patients, but remained nearly unaltered in patients healthy controls and patients with recurrence. These N iu et al. Radiat Oncol (2021) 16:141 Page 11 of 12 OncoRay – National Centre for Radiation Research in Oncology, Faculty with recurrent tumors. Following validation of our data of Medicine and University Hospital Carl Gustav Carus, Technische Universität in a larger patient cohort, monitoring cellular immu- Dresden, Helmholtz‑Zentrum Dresden, Rossendorf, Dresden, Germany. Ger‑ nity in the peripheral blood of SCCHN patients during man Cancer Consortium (DKTK), Partner Site Dresden, Germany. Depar t‑ ment of Radiotherapy and Radiation Oncology, Faculty of Medicine, University therapy might offer an opportunity for an early evalu - Hospital Carl Gustav Carus, Technische Universität, Dresden, Germany. ation of treatment outcome and might help to stratify National Center for Tumor Diseases (NCT ), Partner Site Dresden, Germany. for patients that most likely will benefit from an additive Faculty of Medicine and University Hospital, Partner Site Dresden, Germany. Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. immunotherapy. Helmholtz Association/Helmholtz‑Zentrum Dresden – Rossendorf (HZDR), 17 18 Dresden, Germany. OncoRay, Dresden, Germany. Department of Radiation Oncology, Medical Centre University of Freiburg, Freiburg, Germany. German Abbreviations Cancer Consortium (DKTK), Partner Site Freiburg, Germany. SCCHN: Squamous cell carcinoma of the head and neck; RCT : Radiochemo‑ therapy; CT: Chemotherapy; RT: Radiotherapy; NK: Natural killer cells; NKT: Received: 21 December 2020 Accepted: 19 July 2021 Natural killer‑like T cells; MHC: Major histocompatibility complex. Supplementary Information The online version contains supplementary material available at https:// doi. References org/ 10. 1186/ s13014‑ 021‑ 01868‑5. 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major Additional file 1. Supplementary materials. patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359‑386. 2. Balermpas P, Rödel F, Rödel C, Krause M, Linge A, Lohaus F, et al. CD8+ tumour‑infiltrating lymphocytes in relation to HPV status and clinical Acknowledgements outcome in patients with head and neck cancer after postoperative This work was supported by German Cancer Consortium, DFG (SFB824, chemoradiotherapy: A multicentre study of the German cancer consor‑ STA1520/1‑1, KU3500/2‑1), BMBF (Innovative Therapies 01GU0823, Kompe ‑ tium radiation oncology group (DKTK‑ROG): CD8+ TILs in SCHNN. Int J tenzverbund Strahlenforschung 02NUK038A), BMWi (AiF ZF4320102CS7, Cancer. 2016;138:171–81. ZF4320104AJ8). 3. Stangl S, Tontcheva N, Sievert W, Shevtsov M, Niu M, Schmid TE, et al. Heat shock protein 70 and tumor‑infiltrating NK cells as prognostic Authors’ contributions indicators for patients with squamous cell carcinoma of the head and Patients included in this current study were treated within the HNprädBio neck after radiochemotherapy: a multicentre retrospective study of the trial at 5 DKTK partner sites. 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Journal

Radiation OncologySpringer Journals

Published: Jul 31, 2021

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

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