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Abstract

Objectives: The contemporary concept of carcinogenesis summarizes the role of hypoxia, neoangiogenesis, and hemostasis, including in the stage of progression and metastasis of the tumor process. Metastatic disease is a serious therapeutic challenge for any oncological condition. The purpose of this study was to evaluate the dynamics of specific indicators of neoangiogenesis and hypoxia as potential biomarkers for therapeutic efficacy or risk of disease progression in patients with brain metastases (BM) undergoing robotic stereotactic radiosurgery. Methods: Two groups of patients (lung cancer and other types of cancers) with oligometastatic disease and brain metastases were included. The patients (n=66) were treated with CyberKnife system. Human Angiopoietin-2, Hypoxia inducible factor 1 α (HIF-1α) and human Vascular Endothelial Growth Factor-А (VEGF-А) were measured in this prospective longitudinal study. Results: Analysis of human Angiopoietin-2, HIF-1α, human VEGF-A in the post-treatment period showed a statistically significant decrease between the baseline and the 6 months post-treatment time point in both patient groups. The baseline value of serum VEGF-А in the group with lung cancer decreased by 40%, Аngiopoietin-2-by 48%, HIF-1α -by 43%. In the group with other types of cancers, VEGF-А decreased by 54.75%, Аngiopoietin-2-by 52%, HIF-1α -by 39.5%. Despite the significant reduction, the levels remained significantly higher in both groups than in healthy controls. Conclusion: This study underscores the potential of integrating molecular markers like VEGF-A, Angiopoietin-2, and HIF-1α into clinical decision-making to enhance outcomes for patients with brain metastases undergoing RSRS.

Keywords

solid tumors human Angiopoietin-2 Hypoxia inducible factor 1 (HIF-1α)human VEGF-A brain metastases robotic stereotactic radiosurgery CyberKnife

Introduction

Metastatic disease presents a serious clinical and therapeutic challenge for any oncological condition. Brain metastases (BM) are detected in about 10% of patients at the time of diagnosis.¹ The true incidence of BM, which develops in approximately one-third of patients with carcinoma, is not well known. Lung carcinoma (over 40%), breast carcinoma (about 20%), and melanoma (20%) are the most common primary sites for the development of BM.² The appearance of BM in any oncological localization limits systemic drug therapy due to the pharmacokinetic specificity of medications and the blood-brain barrier. The median survival for untreated patients with BM is about 1–2 months, a period that may increase after the application of local ablative methods such as radiosurgery.

In contrast to the widespread use of molecular-genetic markers in medical oncology, their use for predictive/prognostic purposes in radiotherapy (RT) remains extremely limited. Given the current capabilities of personalized medicine, there is growing interest in combining it with local ablative methods in patients with oligometastatic disease, including BM.³ According to Moravan M. et al, determining the optimal treatment for each specific patient is becoming increasingly individualized, emphasizing the need for multidisciplinary discussion.⁴

The planned scientific study summarizes the contemporary concept of the role of hypoxia and neoangiogenesis in the processes of carcinogenesis, as well as the close relationship between them in the stage of tumor progression and metastasis. Studying these basic indicators would assist in clinical assessment and provide opportunities for individualizing the approach in radiotherapy for patients with BM undergoing robotic stereotactic radiosurgery (RSRS).

The purpose of the present scientific study is to investigate the dynamics of specific indicators of neoangiogenesis and hypoxemia as potential biomarkers for therapeutic efficacy or risk of disease progression in patients with BM undergoing RSRS.

Material and Мethods

A total of 66 oncology patients were treated and monitored during the period 2017–2020. The patients are selected consecutively with confirmed BM as a manifestation of oligometastatic disease (up to 4 metastases) and underwent RSRS. They were divided into two groups: the first group included 34 patients with non-small cell lung cancer (NSCLC). The second group consisted of 32 patients with other types of primary cancer (OTPC) – breast cancer (16), malignant melanoma (9), kidney cancer (4) and colorectal cancer (3). Written informed consent was obtained from all study participants.

The research meets the criteria for scientific ethics of Medical University – Plovdiv and complies with the requirements of: the Declaration of Helsinki on ethics in science; Principle of Good Clinical Practice; Bulgarian laws and regulations for conducting clinical and scientific research involving humans – Opinion №P-9013/15.11.2017. The reporting of this study conforms to STROBE guidelines.⁵ No funding used. All patient details were de-identified.

The patients met the following inclusion criteria: age ≥ 18 years; histologically verified oncological disease; ECOG PS (Eastern Cooperative Oncology Group Performance Status) 1–3; evidence of oligometastatic disease; confirmed BM via MRI – with localization and size meeting the requirements for RSRS. Exclusion criteria were: ECOG PS > 3; a history of heart failure grade III/IV according to NYHA; decompensated diabetes mellitus; acute viral/bacterial infection or exacerbated chronic inflammatory process two weeks before the study; chronic renal failure ≥ grade 2; liver dysfunction – ASAT, ALAT > 5 times the upper reference limit.

The study was designed as a longitudinal prospective study. Before starting therapy (baseline), and at the first, third, and sixth months, patients had follow-up assessments of serum levels of markers for angiogenic activity – human Vascular Endothelial Growth Factor-A (VEGF-A) and angiopoietin-2; markers for hypoxia – hypoxia-inducible factor-1α (HIF-1α); as well as post-therapy monitoring of BM with MRI. In some patients, follow-up was conducted up to the 12th month. The methodology could be reproduced by fellow researchers.

For the purposes of the study, the following methods were used:

1. Detailed Medical Information:

Anamnesis and patient records.

2. Clinical-Laboratory Methods:

2.1. Serum Concentration of Human Angiopoietin-2:

Determined using the ELISA (enzyme-linked immunosorbent assay) heterogeneous immunoenzymatic principle. Ready-made kits from MyBioSource, USA, were used. Analytical reliability: CV within series 5.2%; over time 8.9%; Sensitivity: 10 pg/ml; Analyzer: Multiparameter ELISA Reader - SIRIO, SEAC, Italy.

2.2. Serum Concentration of Human HIF-1α:Principle:

ELISA with ready-made kits from MyBioSource, USA. Analytical reliability: CV within series from 2.97 to 6.0%; over time from 4.12% to 6%; Sensitivity: 0.094 ng/mL; Analyzer: Multiparameter ELISA Reader - SIRIO, SEAC, Italy.

2.3. Serum Concentration of Human VEGF-A:Principle:

ELISA with ready-made kits from Thermo Fisher Scientific, USA. Analytical reliability: CV within series 4.3%; over time 6.2%. Sensitivity: 7.9 pg/ml; Analyzer: Multiparameter ELISA Reader - SIRIO, SEAC, Italy.

3. Control Group:

Serum levels of Angiopoietin-2, human VEGF-A, and Hypoxia Inducible Factor 1 were measured in 30 healthy controls, matched by age and gender to the patient group. All participants were over 18 years old, without any significant comorbidity.

4. Equipment and Protocol for Radiosurgery:

All patients received treatment for local disease control through RSRS using CyberKnife. Based on the size and location of metastases the doses were 1 × 20Gy or 3 × 10Gy. The equipment used had the following specifications:

Digitally controlled linear accelerator for RSRS, CyberKnife M6 FI + System;

Multislice CT “SOMATOM DEFINITION AS 20 Open” - Siemens AG SN: 96453;

Image fusion, tumor volume and critical organ contouring, as well as dosimetric planning, were carried out using the MultiPlan program by Accuray.

5. Statistical Methods:

The selection of statistical methods was aligned with the goals and objectives of the present scientific study, the type of data, and the distribution of variables. Data analysis was performed using the statistical software IBM SPSS, version 26,⁶ the specialized medical analysis software MedCalc version 19.0.7,⁷ and the statistical software Minitab version 19.⁸

Results

Serum Levels of Angiogenic Activity Markers – Angiopoietin-2 and Human VEGF-A

The dynamics of Angiopoietin-2 and human VEGF-A between the baseline and the sixth month were assessed. In both patient groups (NSCLC and OTPC), the values are expressed as medians and interquartile ranges (IQR) due to the presence of asymmetry in the distribution (Kolmogorov-Smirnov P < .05). Intra-group dynamics were tracked using the Friedman test, with post-hoc pairwise comparisons at an adjusted error level of α = 0.012. To more effectively visualize the dynamics, the graphical illustration also includes the values recorded in the control group of 30 healthy individuals.

Figure 1 illustrates the dynamics in VEGF-A and Angiopoietin-2 levels between the baseline and the sixth month. Panel A shows the overall change in VEGF-A levels, indicating a 40% decrease from the baseline level. It is important to note that the VEGF-A level at the sixth month remains significantly higher than that of the healthy controls (187.55 pg/ml), P < .001. The Angiopoietin-2 level (Panel B) decreases by 48% at the sixth month but also remains significantly higher compared to the healthy controls (257.50 pg/ml), P < .001.

Figure 1.

Dynamics in the levels of Angiogenic activity markers VEGF-A and angiopoietin-2 between baseline and the sixth month in the NSCLC group compared to healthy controls.

In patients with OTPC, a similar trend was observed as in the NSCLC group (Figure 2). Both markers showed a comparable pattern of significant reduction in values between the baseline and the sixth month (P < .001). In Panel A, it can be seen that the VEGF-A level decreased by 54.75% at the sixth month, but it still remained significantly higher than the value in healthy controls (187.55 pg/ml), P < .001. The Angiopoietin-2 level decreased by 52% at the sixth month but remained significantly higher than that of the healthy controls (257.5 pg/ml), P < .001 (Panel B).

Figure 2.

Dynamics in the levels of Angiogenic activity markers VEGF-A and Angiopoietin-2 between baseline and the sixth month in the OTPC group compared to healthy controls.

Serum Levels of the Hypoxia Marker – Hypoxia Inducible Factor-1α

The levels of HIF-1α in both patient groups and healthy controls were normally distributed (Kolmogorov-Smirnov P > .05). Statistical analysis was based on the mean values and standard deviation, including one-way analysis of variance (r-ANOVA) and post-hoc pairwise comparisons using the paired t-test with an adjusted error level of α = 0.012.

Figure 3 illustrates a significant decrease in HIF-1α levels between the baseline and the sixth month, with an overall change of 43%. The difference in HIF-1α levels between patients with NSCLC and healthy controls gradually decreased but remained significantly higher in the patients compared to the healthy controls (1.10), P < .001.

Figure 3.

Dynamics in the levels of HIF-1α between baseline and the sixth month in the NSCLC group compared to healthy controls.

Figure 4 shows a significant decrease in HIF-1α levels in patients from the OTPC group between baseline and the sixth month, with an overall change of 39.5%. Despite the gradual reduction in the difference in HIF-1α levels between patients and healthy controls, the HIF-1α value at the sixth month remains significantly higher than that of the healthy controls (1.10), P < .001.

Figure 4.

Dynamics in the levels of HIF-1α between baseline and the sixth month in the OTPC group compared to healthy controls.

Discussion

The theoretical justification for a more aggressive local approach in oligometastatic disease is based on the idea that it represents an “intermediate” state in the evolution of the disease, characterized by a small number of metastases confined to a specific organ.^9,10 This concept, along with the therapeutic role of RSRS as a modern method for local control, was considered in structuring and analyzing the two patient groups. The emergence of BM in any oncological localization complicates and limits treatment options – clinical trials for new medications are challenging due to the short survival of patients (BM are usually an exclusion criteria). There is also unstructured data regarding the potential impact of local radiotherapy methods on neoangiogenesis processes.

The development of BM is facilitated by increased vascular permeability associated with elevated VEGF expression by metastatic cells – this allows easy traversal of the blood-brain barrier. Experimental studies have shown that the rapid growth of primary brain tumors and BM is directly dependent on VEGF expression.¹¹ Suppression of the gene responsible for VEGF production reduces the frequency of BM. The average vascular density at the tumor periphery or within the tumor correlates positively with the aggressiveness of the disease.

Coelho A et al tracked an unselected cohort of 145 patients with NSCLC and investigated serum levels of Angiopoietin-2 and VEGF-A pretreatment.¹² They found statistically significant elevated levels of both neoangiogenesis markers compared to healthy controls. This observation led the team to hypothesize that high level of both Angiopoietin-2 and VEGF-A together, but not VEGF-A alone, could be discussed as a new integrated biomarker for NSCLC patients. Our study confirms these findings. Despite a statistically significant decrease in the values of the neoangiogenesis markers, their levels remain higher than those in the healthy control group.

In the tumor microenvironment, tumor cells and tumor-associated stromal cells (macrophages, other immune cells) produce various angiogenic factors – VEGF, Angiopoietin-2, as well as endogenous inhibitors like thrombospondin.¹³

Hypoxia is a key factor affecting the efficacy of radiotherapy. The introduction of modern radiotherapy machines and the capabilities of RSRS raise questions about the further interpretation of basic knowledge on the subject. In RSRS, reoxygenation is not expected due to severe damage to blood vessels – some tumor zones may remain oxygenated and some hypoxic cells might undergo reoxygenation. This can occur with daily doses below 10 Gy, as vascular damage is significantly less at these doses. At higher daily doses used in RSRS, reoxygenation does not influence outcomes.

Experimental and clinical data demonstrate the role of tumor hypoxia in malignant progression and resistance to radiotherapy.^14,15 Several mechanisms explain the association between tumor hypoxia and poor therapeutic response:^16,17

Complexity of the “dictate” of the hypoxic microenvironment—key factor in tumor resistance, including radioresistance;

Oxygen effect – cells under hypoxia show three times higher radiation resistance compared to normoxic cells;

Selection of resistant clones during carcinogenesis through hypoxia-induced changes in gene expression.

When oxygen levels are high, the content of HIF-1α in cells is very low. However, as oxygen levels decrease, the amount of HIF-1α increases, allowing it to bind and regulate the erythropoietin gene and other genes with HIF-binding DNA segments. Hypoxia protects HIF-1α from degradation.^18,19 The specific oxygen “sensing” allows cells to adapt their metabolism to low oxygen levels. Examples of this mechanism's involvement in physiological processes include embryonic development, immune response, breathing, altitude adaptation, metabolism, and physical activity. Specific oxygen “sensing” is also involved in various pathological conditions—carcinogenesis, neoangiogenesis with active tumor proliferation, anemia, thrombosis (stroke, infarct), infections, and injury and wound therapy.^20,21

The effect of ionizing radiation on angiogenesis is more complex than previously assumed.^22,23 It has been established that there are at least three different pathways through which radiation can affect vascular growth: 1) Protons from electromagnetic radiation partially stimulate vascular growth through increased expression of angiogenic factors; 2) Low linear energy transfer particles, similar to protons, inhibit angiogenesis by unknown mechanisms—thought to reduce angiogenic factor expression and suppress the formation of new blood vessels; 3) High linear energy transfer ions, similar to Fe ions, also provoke inhibition of angiogenesis—the mechanism is still unknown, but the effect occurs in later stages of radiotherapy. The biological effect of radiation increases with linear energy transfer, with higher linear energy transfer resulting in more pronounced biological damage compared to lower linear energy transfer.^20,22,24

Our results align with these fundamental theoretical insights into “hypoxia and radiotherapy”. However, these have not been specifically addressed in the context of RSRS. This is the basis for deepening our research in this direction by monitoring therapeutic responses in our patients with BM. We demonstrated a statistically significant decrease in the hypoxia marker HIF-1α in the post-therapeutic period for both patient groups. It should be emphasized that despite significant changes, HIF-1α levels at the sixth month remained significantly higher in patients compared to healthy controls.²⁵

When interpreting the results and conclusions, it is important to consider the small patient groups that were analyzed. Although the limited number of patients constrains the study, it provides a solid foundation and generates ideas for future research on the topic. On the other hand the observed variations in biomarker responses between NSCLC and other cancer types could be due to differences in tumor biology and angiogenesis mechanisms specific to each type of cancer, or based on the volume of metastases, or based on the fractionation and the effect of treatment.

Conclusion

In contemporary medical literature, studies focusing on a comprehensive analysis of markers characterizing tumor activity in oncological patients undergoing radiotherapy with RSRS are very limited. Our study represents an attempt to make a clinical contribution in this area. Integrating clinical, current laboratory markers and molecular tumor characteristics is promising for making appropriate clinical decisions.

In conclusion, this study reinforces the importance of aggressive local treatment approaches like RSRS in managing oligometastatic disease. By integrating clinical insights with molecular markers, such as VEGF, Angiopoietin-2, and HIF-1α, the research highlights the critical role of neoangiogenesis and hypoxia in therapeutic efficacy or risk of disease progression. Despite significant reductions in neoangiogenesis and hypoxia markers post-treatment, their levels remain elevated compared to healthy controls, indicating persistent tumor activity. This underscores the need for further research to optimize therapeutic strategies, with a particular focus on hypoxia-related resistance mechanisms. Ultimately, the integration of molecular markers into clinical decision-making offers a promising path forward for improving outcomes in patients with BM undergoing RSRS.

Footnotes

Author Contributions

Conceptualization: V.P. and Zh. G-P.; methodology: V.P. and G.R.; validation: V.P.; formal analysis: G.R.; investigation: V.P.; resources: G.R.; data curation: Zh. G-P.; writing—original draft preparation: V.P.; writing—review and editing: V.P., G.R. and Zh. G-P.; visualization: V.P. and G.R.; supervision: G.R.; project administration: Zh. G-P. All authors have read and agreed to the published version of the manuscript.

Data Availability

The data presented in this study are available on request from the corresponding author. The data are not publicly available due privacy restrictions.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Institutional Review Board Statement

The study was performed according to the Declaration of Helsinki, and approved by the Research Ethics Committee of Medical University – Plovdiv (№P-3327//20.12.2017).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

ORCID iD

Veselin Popov

Notes

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