Marco de Bruyn

Background/objectives: Tumor infiltrating lymphocytes (TIL) therapy has yielded promising results in melanoma patients, with an approved product available. TIL therapy is of similar interest to a number of additional malignancies, including endometrial cancer (EC). For EC specifically, the tumor is well infiltrated with TILs and has accessible tumors as starting material for TIL isolation/expansion. However, the TIL manufacturing process currently relies on human serum (HS), which is difficult to source and has considerable batch-to-batch variation. Here, we explored the feasibility of using human platelet lysate (HPL) as a good manufacturing practice (GMP)-compatible alternative. Methods: EC material was obtained following surgery and used to expand TILs in pre–rapid expansion protocols (pre-REP) and REP phases, comparing HPL- and HS-containing media. Tumor tissue from 10 EC patients, representing both high and low tumor mutational burden histotypes was used. Spatial TIL infiltration was assessed using immunohistochemistry. After expansion, TIL phenotype was determined using flow cytometry for CD3, CD56, CD8, CD39, and CD103. TILs from the REP were further characterized with spectral flow cytometry using markers for differentiation, activation, co-stimulatory receptors, and immune checkpoints. Results: TILs expanded comparably using both HPL- and HS-media during pre-REP and REP in terms of cell yields. All tumors showed an excluded spatial immune phenotype. Immunophenotypes were consistent across media conditions. Spectral cytometry revealed patient-specific clustering unaffected by supplement type. Conclusions: TILs can be successfully expanded from EC using a fully GMP-compatible HPL protocol. Our findings may serve as a foundation for further improvements of existing GMP-compliant TIL manufacturing protocols.

Consort diagram and study design. A, Consort diagram depicting the disposition of patients throughout the study and treatment groups. Thirty-eight patients were recruited to this study, of whom 27 were assessable for our analysis. (i) Reasons for exclusion from our analysis were withdrawal of consent, death during the trial, referral to an alternative medical facility, and being subjected to a combination of proton and photon RT regimen. B, Study design. Patients with pathologically and radiologically confirmed stage III–IV HNSCC of the oral cavity, oropharynx, hypopharynx, or larynx were treated with standard-of-care definitive RT with protons or photons (70 Gy) with or without weekly cisplatin. FU, follow-up; HNC, head and neck cancer.

Purpose:Proton therapy delivers more precise radiation (RT) with less toxicity than photons. However, its impact on circulating immune cells in head and neck squamous cell carcinoma (HNSCC) remains poorly understood. We compared the impact of proton versus photon RT on circulating immune cells in patients with locally advanced HNSCC undergoing definitive (chemo)radiation [(C)RT]. Patients and Methods:Patients with stage III to IV HNSCC receiving definitive RT (70 Gy) with or without weekly cisplatin using proton or photon therapy were enrolled. Peripheral blood was collected at baseline, during treatment, and up to 1 year after treatment. White blood cell differentiation, lymphocyte subsets, and lymphocyte function were evaluated using high-dimensional flow cytometry and functional assays. Results:Twenty-seven patients were enrolled: Ten received CRT with protons, eight received RT alone with protons, and nine received RT alone with photons. Absolute lymphocyte counts and naïve CD4+ T cells declined in all groups, whereas NK cells increased and memory T-cell populations remained largely unaffected, with no additional effect of concurrent cisplatin. Lymphocyte suppression persisted for up to 6 months in the CRT proton group, 3 months in the RT proton group, and 12 months in the RT photon group. Greater low-dose body irradiation (2 Gy) was correlated with lower lymphocyte counts at 5 weeks and 6 months after treatment and was more common in photon-treated patients. Conclusions:Both proton and photon RT markedly suppress circulating lymphocytes, particularly naïve CD4+ T cells, for at least 3 months after treatment in locally advanced HNSCC. Significance:Proton RT is increasingly utilized for head and neck cancer. However, its impact on systemic immunity remains unclear. We demonstrated that both proton and photon RT caused prolonged suppression of circulating lymphocytes for at least 3 months. Naïve CD4+ T cells were preferentially depleted, whereas memory T-cell populations were largely spared. These findings highlight the importance of considering immune effects and minimizing off-target lymphocyte exposure when combining radiotherapy with immunotherapy.

Absolute number of circulating immune cells at baseline, during, and after RT (up to 12 months after completion of treatment) using protons compared with photons. A–D, Tukey box plots showing the (A) leukocyte counts, (B) lymphocyte counts, (C) neutrophil counts, and (D) monocyte counts in patients treated with CRT with protons, RT with protons alone, and RT with photons alone at baseline, during treatment, and following treatment from 2 weeks up to 12 months after treatment. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. T1, visit 1 (baseline); T2, visit 2 (during treatment); T3–7, visits 3–7 (follow-up visits at 2–52 weeks after completion of treatment, respectively). The symbols (triangle, square, and circle) in the figure represent outliers.

RT dose and body volume in relation to absolute lymphocyte counts. A and B, Representative RT therapy plan for a patient with HNSCC in the study treated with (A) proton therapy and (B) photon therapy. C–F, Tukey box plots showing the differences in (C) CTV receiving a boost dose (70 Gy), (D) CTV receiving an elective treatment dose (54.25 Gy), (E) dose administered to the area outside of the CTV spanning from the sphenoid sinus to the sternum, and (F) dose administered to the vertebral bodies of the cervical spine among patients subjected to CRT with protons, RT with protons alone, and RT with photons alone. G and H, Scatterplots showing the correlation between the percentage of body volume receiving 2 Gy of RT and absolute lymphocyte counts at (G) T4 and (H) T6. I and J, Scatterplots showing the correlation between the percentage of body volume receiving 0.5 Gy of RT and absolute lymphocyte counts at (I) T4 and (J) T6. K and L, Scatterplots representing the correlation between RT dose administered outside of the CTV and absolute lymphocyte counts at (K) T4 and (L) T6. M and N, Scatterplots representing the correlation between RT dose administered to the vertebral bodies of the cervical spine and absolute lymphocyte counts at (M) T4 and (N) T6. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. ρ, Spearman correlation coefficient. The dotted line denotes the lower boundary for absolute lymphocyte counts. T4 and T6, visit 4 and 6, follow-up visits at 5 weeks and 6 months after completion of treatment, respectively. cGy, centigray; RBE, relative biological effectiveness.

Correlations between body and organ-specific radiation exposure and lymphocyte counts.

Overview of the characteristics of the seven identified clusters significantly changing upon radiotherapy.

Lymphocyte subsets affected by proton and photon RT. A–C, Box plots with whisker plots depicting the increase in relative abundance of NK cells (CD3−CD56+CD16+CD57+CD27−CD127−CD45RA+) during and after radiotherapy, represented by clusters (A) 729994, (B) 729996, and (C) 729998. D–G, Box plots with whisker plots depicting the significant decrease in the relative abundance of naïve CD4+ T cells (CD3+CD4+CD45RA+CCR7+CD27+CD25−CD127+CD38−CD16−CD57−) during and after radiotherapy, represented by clusters (D) 729987, (E) 729992, (F) 729993, and (G) 729997. T1–6, visit 1–6 [baseline, during treatment, and follow-up visits at 2–26 weeks (i.e., 6 months) after treatment completion, respectively].

Memory T-cell functionality during and after radiotherapy with protons versus photons. A and B, Tukey box plots showing the capacity of T cells to respond to (A) CEF peptides and (B) COVID peptides during and following treatment (up to 6 months after treatment). CEF, 23 different viral peptides of human cytomegalovirus; COVID, 315 peptides derived from the Spike glycoprotein of SARS-CoV-2; EBV, Epstein–Barr virus; flu, influenza A virus; T1–6, visits 1–6 [baseline, during treatment, and follow-up visits at 2–26 weeks (i.e., 6 months) after treatment completion, respectively].

Memory T-cell functionality to stimulation with CEF and Covid peptides per patient during and after radiotherapy using protons versus photons.