To perform the analysis of the peripheral blood lymphocyte changes after stereotactic ablative radiotherapy (SABR) in patients with oligometastatic cancers.
The dynamics of the immune status in peripheral blood was prospectively evaluated in 46 patients with lung (17 cases) or liver (29 cases) metastases treated by SABR. Flow cytometry of peripheral blood lymphocyte subpopulations was performed before SABR, 3–4 weeks and 6–8 weeks after the end of SABR: 3 fractions of 15–20 Gy or 4 fractions of 13.5 Gy. The number of treated lesions varied from 1 (32 patients) to 2–3 (14 patients).
SABR induced a significant increase of T-lymphocytes (CD3+CD19–) (p = 0.001), T-helper (CD3+CD4+) (p = 0.004), activated cytotoxic T-lymphocytes (CD3+CD8+HLA-DR+) (p = 0.001), activated T-helpers (CD3+CD4+HLA-DR+) (p < 0.001). A significant decrease of T-regulated immune suppressive lymphocytes (CD4+CD25brightCD127low) (p = 0.002) and NKT-cells (CD3+CD16+CD56+) (p = 0.007) was recorded after the SABR. The comparative analysis demonstrated that lower doses of SABR (EQD2Gy(α/β=10) = 93.7–105.7 Gy) induced significant increase of T-lymphocytes, activated cytotoxic T-lymphocytes, and activated CD4+CD25+ T-helpers, while SABR with higher doses (EQD2Gy(α/β=10) = 150 Gy) was not associated with these effects. A more efficient activations of T-lymphocytes (p = 0.010), activated T-helpers (p < 0.001), and cytotoxic T-lymphocytes (p = 0.003) were associated with SABR to a single lesion. A significant increase of T-lymphocytes (p = 0.002), T-helpers (p = 0.003), and activated cytotoxic T-lymphocytes (p = 0.001) was observed after SABR for hepatic metastases in contrast to SABR for lung lesions.
Changes in peripheral blood lymphocytes after SABR could be influenced by the location or the number of irradiated metastasis, and the dose of SABR.
Radiotherapy is an important and obligatory component of treatment programs in 50%–70% patients with cancer [
A certain number of hypotheses tried to explain systemic effects of radiotherapy. It was proposed that the radiation damage of various tumors is associated with the release of increased presentation of antigens and release of effector molecules (HMGB1) triggering molecular patterns associated with damage of tumor cells (DAMPs), activation of proteasomal pathways and major histocompatibility complex molecules (HLA) of the 1st class, NKG2D ligand expression, complement fixation, interferon 1st type production, and induction of cell immunogenic death [
Unfortunately, until now the immunologic changes occurring during and after the SABR have not been sufficiently studied in clinical practice. In the presented prospective observation study, we have performed a complex analysis of the dynamics of immune status before and after SABR of various (in number and localization) metastatic lesions.
The dynamics of immune status in peripheral blood was prospectively evaluated in 46 patients (30 females and 16 males; mean age, 55.9 years; 95% CI, 52.4–59.4) with lung or liver metastases treated by SABR from September 2018 to July 2021.
This study was prospective observation single center study, and it was approved by the local ethics committee of N.N. Petrov National Cancer Center (Protocol No. 13, dated November 29, 2018). Voluntary informed consent for stereotactic radiotherapy and immunological studies was obtained in writing form from all patients.
Inclusion criteria were as follows: morphologically confirmed disease; oligometastatic disease (less than four lesions); the first SABR session; anatomical localization of metastases in liver or lungs, allowing for a radical SABR course; the size of liver or lung metastasis is less than 5 cm; no treatment by immune checkpoints inhibitors or/and chemotherapy at least 2 months before and after SABR.
Exclusion criteria were radiotherapy 6 or less months before SABR; immunotherapy before or during the treatment; age <18 years; Karnofsky performance status <70%; chemotherapy 2 months before or during SABR.
The immune status of patients was evaluated before irradiation, 3–4 and 6–8 weeks after the end of SABR. Distribution of the patients according to the localization of the primary lesion and irradiated metastatic sites is presented in
SABR was performed according to the standard volumetric modulated arc therapy technique with respiratory gating and obligatory cone-beam CT guidance of every session. Lung metastases were irradiated as 3 fractions of 20 Gy (peripheral metastases) or 4 fractions of 13.5 Gy (lesions with proximity to ribs or central “fly zone”). Liver metastases were irradiated in 3 fractions of 15–20 Gy. The number of treated lesions varied from 1 (32 patients) to 2–3 (14 patients). The dynamics of immune status was analyzed in patients with liver (29 cases) and lung (17 cases) metastases separately after SABR within the equivalent dose—EQD2Gy(α/β=10) of 93.75–105.75 Gy (group A, 23 patients) or EQD2Gy(α/β=10) of 150 Gy (group B, 23 patients). All included patients underwent SABR treatment for all oligometastatic lesions.
The assessment of immune status was made in three checkpoints: before the radiation (checkpoint 1), in 3–4 weeks (checkpoint 2), and in 6–8 weeks (checkpoint 3) after the end of SABR. The immune status was characterized by the count of several main peripheral blood lymphocyte subpopulations.
The calculations were performed with flow cytometry method. To process the data, we used BD FACSDiva software (version v8.0.1; BD Biosciences, San Jose, CA, USA). The subpopulation composition of peripheral blood immunocompetent cells was studied on a BD FACSCanto II flow cytometer. We evaluated the relative value of lymphocytes; T-lymphocytes (CD3+CD19–); В-lymphocytes (CD3–CD19+); T-helpers (CD3+CD4+); activated T-helpers with different phenotypes (CD3+CD4+HLA-DR+ and CD4+CD25+); cytotoxic Т-lymphocytes (CD3+СD8+); activated cytotoxic Т-lymphocytes (CD3+СD8+HLA-DR+); NK-cells (CD3–CD16+CD56+); NKТ-cells (CD3+CD16+CD56+); T-regulatory lymphocytes (CD4+CD25brightCD127low); double-positive T-lymphocytes (CD3+CD4+СD8+); double-negative T-lymphocytes (CD3+CD4–СD8–); and the immunoregulatory index (CD4+/CD8+).
The values of this immune status indicators (T-lymphocytes; В-lymphocytes; T-helpers; activated T-helpers with phenotype CD3+CD4+HLA-DR+; cytotoxic Т-lymphocytes; activated cytotoxic Т-lymphocytes; NK-cells; NKТ-cells; double-positive T-lymphocytes; double-negative T-lymphocytes and the immunoregulatory index) were calculated from the total count of lymphocytes. CD4+CD25+ activated T-helpers and T-regulatory lymphocytes were calculated as a percentage of the T-helper (CD3+CD4+) population.
For calculation of the absolute value of the immunocompetent cells, a two-platform analysis system was used; the calculation was made using the results of a hematological analyzer.
We used antibodies for a multicolor panel (BD FACSCanto2 HIV reagents kits, 6-color TBNK reagent) mouse anti-human HLA-DR monoclonal antibodies labeled with V450, CD25 antibodies labeled with FITC (BD Biosciences), CD4 monoclonal antibodies labeled with BD PerCP-Cy5.5 fluorochrome and CD127 antibodies labeled with Pe-Cy7.
The statistical analysis of the data was performed in R version 3.6.2 (
The analysis of immune status parameters in patients with extensive irradiated solid tumors revealed a statistically significant increase of T-lymphocytes (CD3+CD19–) in 3–4 weeks after the irradiation compared to the analysis performed before the stereotactic radiotherapy (
A statistically significant increase of activated cytotoxic T-lymphocytes carrying the HLA-DR+ antigen was noted in 3–4 weeks after SABR (pairwise p = 0.001) with a continuing rise of this subpopulation in 6–8 weeks after the irradiation (pairwise p = 0.004) (
An increase of activated T-helpers was noted 3–4 and 6–8 weeks after the SABR compared to the values obtained before the irradiation (
A decrease of T-regulated immune suppressive lymphocytes (CD4+CD25brightCD127low) was recorded in 3–4 and 6–8 weeks after the SABR (
Besides, a decrease of B-lymphocytes (CD3–CD19+) was recorded in 3–4 and 6–8 weeks after the SABR compared to the values recorded before the irradiation (
A decrease of NKT-cells (CD3+CD16+CD56+) was noted in 6–8 weeks after the irradiation compared to the analysis performed before the SABR (
Statistically significant differences in the dynamics of other immune cells in the peripheral blood (activated CD4+CD25+ T-helpers, NK-cells, double positive T-lymphocytes, double negative T-lymphocytes) were not revealed.
The comparative analysis demonstrates a significant correlation between changes in the peripheral blood immune cells and the EQD2Gy(α/β=10) delivered to the metastatic lesions. While examining T-lymphocytes (CD3+CD19–) after the SABR, a statistically significant increase of T-lymphocytes was discovered in 3–4 weeks after the irradiation in both dose groups—group A: EQD2Gy(α/β=10) = 93.75–105.75 Gy (
The dynamic pattern in activated cytotoxic T-lymphocytes (CD3+CD8+HLA-DR+) is quite interesting. When administering EQD2Gy(α/β=10) = 93.75–105.75 Gy, a statistically significant increase of cells in the given population was noted both in 3–4 and 6–8 weeks after the irradiation (
A statistically significant increase of T-helpers was recorded in 3–4 weeks after irradiating the lesions with EQD2Gy(α/β=10) = 93.75–105.75 Gy (
A statistically significant increase of activated T-helpers phenotype (CD3+CD4+HLA-DR+) was noted in 3–4 and 6–8 weeks after the SABR when compared to the values obtained before SABR. These differences were significant in both groups: in group A (EQD2Gy(α/β=10) = 93.75–105.75 Gy) (
Moreover, the analysis of another subpopulation of the activated T-helpers (CD4+CD25+) demonstrated a statistically significant increase of activated T-helpers in 6–8 weeks after the SABR within the EQD2Gy(α/β=10) = 93.75–105.75 Gy (group A) when compared to the values obtained before the SABR (
In the subgroup of T-regulatory (suppressors) lymphocytes we also found dose dependent effects. In particular, a significant reduction of T-regulatory lymphocytes (
In 46 consecutive patients, we compared the dynamics of immune status according to the number of the irradiated lesions. Group I consisted of 32 patients with one irradiated metastatic lesion and group II (14 patients) with 2–3 irradiated metastases. After SABR of a single lesion we mentioned numerous changes in immune profile. First of all, a statistically significant increase of T-lymphocytes (CD3+CD19–) was recorded in 3–4 weeks after the SABR (
The increase of activated T-helpers (CD3+CD4+HLA-DR+) was noted in group I in 3–4 weeks and 6–8 after the irradiation compared to the values obtained before the SABR (
A statistically significant increase of the cytotoxic T-lymphocytes was revealed in 3–4 and 6–8 weeks after the SABR of a single lesion (
The analysis of T-regulatory lymphocytes in group with one irradiated lesion demonstrated a decrease of this subpopulation in 3–4 and 6–8 weeks after SABR compared to the values detected before the start of radiotherapy (
We also evaluated changes of the immune status in connection with the localization (lung vs. liver) of irradiated metastases. An increase of activated T-helpers was observed in both groups 3–4 and 6–8 weeks after SABR when compared to the values obtained before the irradiation: in group with SABR for hepatic lesions (
Significant changes in the number of T-lymphocytes (
A statistically significant increase of activated cytotoxic T-lymphocytes carrying antigen HLA-DR+ was registered in 3–4 weeks (pairwise p = 0.004) and in 6–8 weeks (pairwise p = 0.010) (
Moreover, a statistically significant increase of the immunoregulatory index was noted 3–4 weeks after SABR for liver metastases (pairwise p = 0.040) followed by a decrease in 6–8 weeks after the irradiation (
On the contrary, a statistically significant decrease of T-regulatory lymphocytes (
Different populations of immune cells characterizing SABR induced changes of the immune status were dynamically evaluated during this study. These parameters reflect differences in both T-cell and humoral component of the immune system. The obtained results identify the activation of T-cell immune response mainly by the increase of T-lymphocytes (CD3+CD19–); T-helpers (CD3+CD4+); activated T-helpers (CD3+CD4+HLA-DR+) and activated cytotoxic T-lymphocytes (CD3+СD8+HLA-DR+) against the decrease in activity of the antibody producer cells (CD3–CD19+).
Chua et al. [
Other authors [
Considering the above-mentioned issues, a special focus in interpretation of our research must be concentrated on the post-SABR activation of T-cell component: T-lymphocytes (CD3+CD19–), T-helpers (CD3+CD4+), activated T-helpers (CD3+CD4+HLA-DR+), and activated cytotoxic T-lymphocytes (CD3+СD8+HLA-DR+) with parallel reduction in the number of T-regulatory lymphocytes. These changes indicated that the state of T-cell inflammatory phenotype or “hot” tumor is likely to be reached after SABR. As is proved by Turajlic et al. [
Most of the discussions about the possible additive or supra-additive effect of immune check point inhibitors with irradiation underline an important role of proper sequencing of these modalities. In particular, Buchwald et al. [
The effect of radiotherapy technique and the value of absorbed dose on the antitumor immune response intensity is still a matter for debates. Some authors reported that irradiation with large (>10 Gy) dose per fraction and high absorbed radiation dose causes massive release of tumor antigens and increased frequency of abscopal effects [
Many authors [
These conflicting results can be partly explained by the fact that in preclinical and clinical studies, response to immunotherapy has been shown to be strongly associated with tumor burden, with higher response in lower burden [
Currently, there is no clear understanding of what kinds of immune system changes occur in metastatic patients after SABR administered to lesions of various localizations. Tang et al. [
Our study has a number of meaningful limitations. First, it is a relatively small number of evaluated patients that might have underpowered some changes in the immune landscape (as in irradiating lung lesions or in SABR to multiple lesions). Moreover, the obtained data might have been critically impacted by the group heterogeny with regard to the localization and histological variant of the primary tumors. In order to overcome these limitations we tried to perform multivariate analysis, but this has not been successful due to a very small sample size of the subgroups.
However, it seems possible to make several preliminary conclusions: (1) SABR is associated with the activation of T-cell component of the immune system (p ≤ 0.01) which is manifested by the increase of T-lymphocytes (CD3+CD19–), T-helpers (CD3+CD4+), activated T-helpers (CD3+CD4+HLA-DR+), and activated cytotoxic T-lymphocytes (CD3+СD8+HLA-DR+) affected by the decrease of regulatory T-lymphocytes (CD4+CD25brightCD127low) and antibody producer cells (CD3–CD19+). The activation is most evident in 3–4 weeks after the SABR. (2) Lower doses of SABR (EQD2Gy(α/β=10) = 93.7–105.7 Gy) are associated with the most significant activation of T-cell immune response (p ≤ 0.04) than SABR with higher total dose (EQD2Gy(α/β=10) = 150 Gy). (3) SABR for a single metastatic lesion induces a more pronounced activation of T-cell immune response than irradiation of two or three lesions (p ≤ 0.03). (4) SABR for a hepatic lesion result in a more pronounced activation of T-cell immune response than SABR for lung metastases (p ≤ 0.04).
Immunogenic effects of radiation therapy are complex, controversial and not perfectly understood. It becomes clear that many factors (localization and number of irradiated lesions, the dose and fractionation scheme, irradiated volume, etc.) can significantly influence the immunomodulating activity of radiation therapy, and further research in this field is of paramount clinical importance.
This study was approved by the local ethics committee of N.N. Petrov National Cancer Center (Protocol No. 13 dated November 29, 2018). Voluntary informed consent for stereotactic radiotherapy and immunological studies was obtained in writing form from all patients.
No potential conflict of interest relevant to this article was reported.
None.
Conceptualization, Novikov SN, Aleksandrovna BI, Vasilevich KS. Formal analysis, Novikov SN, Aleksandrovna BI, Yurievich ZA. Data curation, Viktorovich GD, Viktorovich GD, Ivanovna TE, Evgenevich AF, Ivanovich AA, Markovich GM. Investigation and methodology, Viktorovna EN. Writing–original draft, Novikov SN, Yurievich ZA. Writing–review and editing, Novikov SN, Aleksandrovna BI, Yurievich ZA, Viktorovna EN, Viktorovich GD, Alexandrovna FE, Ivanovna TE, Evgenevich AF, Ivanovich AA, Markovich GM, Vasilevich KS.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supplementary materials can be found via
Statistical data of immune status indicators in the studied points against the background of SABR
Statistical data of immune status indicators in the studied points against the background of SABR depending on delivered EQD2Gy(ɑ/β=10)
Statistical data of immune status indicators in the studied points against the background of SABR depending to the number of the irradiated lesions
Statistical data of immune status indicators in the studied points against the background of SABR depending to localization of irradiated metastases
Dynamics in the number of (A) activated cytotoxic lymphocytes, (B) activated T-helpers, and (C) T-regulatory lymphocytes. SABR, stereotactic ablative radiotherapy.
Dynamics in the number of (A) activated cytotoxic lymphocytes, (B) activated T-helpers, and (C) T-regulatory lymphocytes depends on delivered EQD₂Gy (ɑ/β = 10). SABR, stereotactic ablative radiotherapy; EQD, equivalent dose.
Dynamics in the number of (A) T-helpers, (B) activated T-helpers, and (C) T-regulatory lymphocytes according to the number of the irradiated lesions. SABR, stereotactic ablative radiotherapy.
Dynamics in the number of (A) T-helpers, (B) activated cytotoxic lymphocytes, and (C) T-regulatory lymphocytes according to localization of irradiated metastases. SABR, stereotactic ablative radiotherapy.
Patient distribution according to the primary tumor and localization of the irradiated metastases
Primary | n | Localization of the irradiated metastases |
|
---|---|---|---|
Liver | Lung | ||
Colorectal adenocarcinoma | 20 | 17 | 3 |
Breast cancer | |||
Luminal B subtype | 5 | 4 | 1 |
HER2-enriched subtype | 1 | 0 | 1 |
Triple-negative subtype | 1 | 1 | 0 |
Non-small cell lung cancer | |||
Adenocarcinoma | 1 | 0 | 1 |
Squamous cell carcinoma | 2 | 1 | 1 |
Adenosquamous carcinoma | 1 | 1 | 0 |
Poorly differentiated | 1 | 0 | 1 |
Soft tissue sarcoma | |||
Leiomyosarcoma | 4 | 2 | 2 |
Extraskeletal myxoid chondrosarcoma | 1 | 0 | 1 |
Parotid cancer | |||
Adenoid cystic carcinoma | 1 | 0 | 1 |
Clear cell carcinoma | 1 | 0 | 1 |
Low grade adenocarcinoma | 1 | 0 | 1 |
Epithelioid malignant melanoma | 2 | 2 | 0 |
Esophageal squamous cell carcinoma | 1 | 1 | 0 |
Prostate acinar adenocarcinoma | 1 | 0 | 1 |
Hepatocellular carcinoma | 1 | 0 | 1 |
Clear cell renal cell carcinoma | 1 | 0 | 1 |
Total | 46 | 29 | 17 |
HER2, human epidermal growth factor receptor 2.