Research ArticleExperimental Studies
Open Access

Dose Distribution Degradation of Carbon-ion Radiotherapy Caused by Tumor Cell-specific Relative Biological Effectiveness of Osteosarcoma: A Simulation Study Using In Vitro Experimental Results

SHINTARO SHIBA, MOTOHIRO KAWASHIMA, MASAHIKO OKAMOTO and TATSUYA OHNO
Anticancer Research November 2023, 43 (11) 4873-4878; DOI: https://doi.org/10.21873/anticanres.16684

Abstract

Background/Aim: Dose distributions of carbon-ion radiotherapy (C-ion RT) have been created with the relative biological effectiveness (RBE) of human salivary gland cells (HSG). However, no dose distributions have been created using various tumor cell-specific RBE values. Hence, we conducted in vitro experiments to determine the RBE of human osteosarcoma cells (U2OS) and used this RBE value (RBEU2OS) to calculate the dose distribution for C-ion RT. Materials and Methods: To obtain RBE values for various linear energy transfer (LET) levels, we exposed U2OS cells to different doses of X-rays and varying doses and LET levels of C-ion beams (13, 30, 50, and 70 keV/μm). Subsequently, we converted the RBE of HSG (RBEHSG) to RBEU2OS in the treatment planning system and reconstructed the dose distribution for a typical osteosarcoma case. We performed a dose-volume histogram (DVH) analysis, evaluating the percentage of the minimum dose that covered 98%, 50%, and 2% (D98%, D50%, and D2%, respectively), as well as the homogeneity index [HI; calculated as (D2%-D98%)/D50%]. Results: The RBEU2OS values for C-ion beams with LET of 13, 30, 50, and 70 keV/μm were 1.77, 2.25, 2.72, and 4.50, respectively. When comparing DVH parameters with the planning target volume, we observed the following values: D98%, D50%, D2%, and HI for RBEHSG were 64.1, 70.1, 72.4 Gy (RBE), and 0.12, respectively. For RBEU2OS, these values were 86.2, 95.0, 107.9 Gy (RBE), and 0.23, respectively. Conclusion: We utilized RBEU2OS to calculate the dose distribution of carbon ion radiotherapy, revealing potential degradation in dose distribution and particularly worsening of the HI.

Key Words:

Carbon-ion radiotherapy (C-ion RT) was initiated in 1994 to treat various cancers in Japan, and favorable clinical results have been reported (1-5). These results are due to its higher dose localization property by the C-ion beam characteristics, including the Bragg peak, sharp lateral penumbra, and higher relative biological effectiveness (RBE) due to high linear energy transfer (LET) in the Bragg peak compared to X-ray RT (1). Furthermore, C-ion beams with high LET exhibit a superior cell-killing effect compared to X-rays, even for radioresistant tumors such as sarcomas (3, 6).

The clinical C-ion RT dose is calculated based on the physical dose multiplied by the RBE of the C-ion beams and is expressed in Gy (RBE) (7). The clinical RBE was calculated using experimental results of survival response after C-ion beam irradiation on several cell types, including human salivary gland (HSG) cells. Consequently, the clinical RBE of the C-ion beam was determined to be 3 under irradiation conditions with a 6 cm spread-out Bragg peak (SOBP) of 290-MeV/u C-ion beams; however, the experiments to obtain this RBE did not involve sarcoma or radioresistant tumor cells (8). Therefore, different doses may be prescribed for sarcomas in clinical practice. In particular, the prescribed dose point is central to the SOBP in clinical practice. If the RBE at the center, distal side, and proximal sides of the SOBP of the HSG and sarcoma cells are relatively different, the dose distribution may not be uniform and could be compromised. Additionally, tumor-specific dose distributions have not been created using sarcoma cells, and there are no reports on whether dose distributions are affected. Hence, we determined the dose distribution using the RBE calculated from the survival response of human osteosarcoma cells (U2OS) and compared the dose-volume histogram (DVH) parameters between the HSG-based and U2OS-based dose distributions using a clinical sarcoma case.

Materials and Methods

Cell line. The U2OS cells used in this study were obtained from ATCC® (HTB-96™). Cells were maintained on 10-cm tissue culture plates at 37°C in a humidified atmosphere with 5% CO2 using Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. The medium and serum were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Cells were passaged before reaching confluence and were used for all experiments within 10 passages after purchase from JCRB to ensure stable results.

X-ray and C-ion beam irradiation. X-ray irradiation was performed using an MX-160Labo (MediXtec, Matsudo, Japan) at Gunma University. The dose rate and energy of X-ray irradiation were 1.06 Gy/min and 160 keV, respectively. The cells were irradiated with different doses of radiation (2, 4, 6, and 8 Gy). C-ions were accelerated to 290 MeV/nucleon using a Heavy-ion Medical Accelerator at Gunma University Heavy Ion Medical Center. The irradiation system and biophysical characteristics have been reported previously (9). The cells were irradiated with different doses (1, 2, 3, and 4 Gy) at LET values of 13, 30, 50, and 70 keV/μm for the C-ion beams. All experiments were performed at least three times.

Clonogenic cell-survival assay. The effect of the treatment on cell survival was evaluated using a clonogenic cell survival assay. Cells were seeded into six-well tissue culture plates and exposed (or not) to X-ray or C-ion beam irradiation. After incubation for 10-14 days, the cells were fixed with methanol and stained with crystal violet. Colonies containing at least 50 cells were counted. The survival fractions were calculated as the ratio of surviving colonies to the number of plated cells. Surviving cell fractions were normalized to the surviving fraction without irradiation (control). The dose resulting in a surviving fraction of 10% (D10) was calculated using a linear quadratic model (LQ model) (10). The RBE values were obtained by comparing the D10 values for both C-ion beams and X-rays. Additionally, we determined the LET dependency of the RBE curve in U2OS cells based on the results of in vitro experiments and the LET dependency of the RBE curve in HSG cells.

Conversion of RBE on treatment plan and DVH analysis. We scaled the RBE calculated using the HSG (RBEHSG) to complement the U2OS (RBEU2OS) experimental results. Next, we converted the RBEHSG to RBEU2OS and reconstructed the dose distribution in a typical case of osteosarcoma. Target delineation, prescribed dose calculated by the RBEHSG, planning aim, and dose constraint of osteosarcoma cases have been previously reported (6). Subsequently, we assessed the percentage of the minimum dose that covered 98%, 95%, 50%, and 2% of the target volume (D98%, D95%, D95%, and D2%), the percentage of target volume irradiated with 60-110 Gy (RBE) (V60-110Gy(RBE)), and the homogeneity index [HI; (D2%-D98%)/D50%] based on the DVH for the gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV). This DVH analysis was approved by the Institutional Review Board (approval number: HS2019-130).

Results

Relative biological effectiveness. The survival curves under different irradiation schemes in U2OS cell lines are shown in Figure 1. D10 of C-ion beams with LET of 13, 30, 50, in 70 keV/μm, and X-rays were 3.60 Gy, 2.83 Gy, 2.34 Gy, 1.41 Gy, and 6.38 Gy, respectively. The RBEU2OS values of C-ion beams with LET of 13, 30, 50, and 70 keV/μm were 1.77, 2.25, 2.72, and 4.50, respectively. Additionally, we used these results to create RBEU2OS curves by linearly complementing the experimental results and scaling the RBEHSG to a higher LET where there were no experimental results. Table I shows the RBEHSG in the treatment planning system of C-ion RT and RBEU2OS resulting from the experiment for each LET.

Figure 1.
Figure 1.

Survival curves of U2OS cells after X-ray (A) or carbon-ion beam irradiation with linear energy transfer (LET) of 13 keV/μm (B), 30 keV/μm (C), 50 keV/μm (D), and 70 keV/μm (E). Data are presented as mean±standard deviation, fitted to the linear-quadratic model.

View this table:
Table I.

Relative biological effectiveness (RBE) values for HSG and U2OS cells.

Reconstruction of dose distribution and DVH analysis. We recalculated the clinical dose using the RBEU2OS and reconstructed the dose distribution in the osteosarcoma case (Figure 2). Comparing the DVH parameters, the HI in GTV calculated by RBEHSG to RBEU2OS changed from 0.05 to 0.20, those in CTV changed from 0.06 to 0.22, and those in PTV changed from 0.12 to 0.23, respectively. Other parameters are shown in Table II.

Figure 2.
Figure 2.

Dose distribution on an axial computed tomography image. The area within the red, cyan, and yellow outline is gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV), respectively. (A) Dose distribution calculated by relative biological effectiveness (RBE)HSG. (B) Dose distribution calculated by RBEU2OS.

View this table:
Table II.

Comparison of dose-volume histogram (DVH) parameters calculated by RBEHSG and RBEU2OS.

Discussion

We reconstructed the dose distributions with RBEU2OS and showed dose distribution degradation with HI deterioration for the GTV, CTV, and PTV. Additionally, we found that this degraded dose distribution may differ from the dose distribution intended by the radiation oncologist in that the dose distribution was not uniform. This non-uniform tumor-specific dose distribution may be the cause of recurrence.

For C-ion RT, dose-escalation studies have been conducted for each disease to establish the recommended total dose (11, 12). These recommended doses were calculated using the RBEHSG. In contrast, dose distribution was calculated using RBEU2OS in this study. This study does not propose changing the current therapeutic doses because the current dose calculation using the RBEHSG showed favorable treatment outcomes. Additionally, although a Dmax of 111.7 Gy (RBE) calculated by RBEU2OS appears to be an excessive dose, the physical dose is no different from that used in the real plan (calculated by RBEHSG), and considering that the 5-year local control rate of C-ion RT for bone and soft tissue sarcomas is approximately 70% (3, 6, 11, 13), this does not mean that the prescribed dose should be lowered. Since the adverse events are tolerable, we believe it is appropriate to continue C-ion RT at the prescribed dose. However, our suggestion is to generated awareness that the dose in the tumor may be a gradient rather than uniform, as calculated by RBEU2OS, and that there may be some areas where the dose is lower than the radiation oncologist expected, especially in low-LET areas. Future studies should analyze recurrent cases of bone and soft tissue sarcomas and confirm whether recurrence occurs in areas with low tumor-specific doses.

Our in vitro experiments showed that the RBE in the higher LET group had a higher rate of increase than that in the lower LET group, which also affected the reconstruction of the dose distribution calculated using RBEU2OS. Therefore, the difference between D98% and D2% widened, and dose distribution degradation occurred with a worsening HI. In some cases, especially in the current case, the reconstruction of the dose distribution with a tumor cell-specific RBE value can result in lower doses on the proximal side of the beam path than on the distal side, which is caused by heterogeneous LET distributions. Therefore, when determining the beam directions, it is necessary to be aware of the LET distribution and plan to make the LET distribution as uniform as possible (e.g., using a beam angle such that the beam paths on the proximal side do not overlap).

The LQ model is valuable for predicting the biological effects of RT, such as tumor control and normal tissue toxicity (14). Additionally, it enables the comparison of different types of radiation, such as X-rays and C-ions, by quantifying their RBE. This RBE information would be useful in selecting treatment. However, the LQ model had several limitations. First, although the LQ model assumes uniform sensitivity of tissues to RT, tissues have varying sensitivities in practical terms. Second, in cases of C-ion RT, the radiobiology is more complex than that of X-rays (15). This complexity is not fully captured by the LQ model. Third, the LQ model may not accurately predict the effects of extreme hypofractionation or very high doses per fraction (16). Thus, to date, the biological perspective for predicting RBE during clinical practice with C-ion RT remains debatable.

Permata et al. calculated the RBEU2OS of C-ion beams at 60 keV/μm as 3.24 (17). Although we did not irradiate U2OS cells at 60 keV/μm in this study, their finding is between ours at 50 and 70 keV/μm, and we believe our cell irradiation results are reasonable.

It is currently impossible to calculate tumor cell-specific RBE values and create dose distributions using a patient’s tumor cells during treatment because of cell culture and time constraints. However, we believe that even if the RBE is not calculated using patient-specific tumor cells, confirming the dose distribution created by the RBE for each cancer type may improve treatment outcomes.

This study has several limitations. First, a limited number of LET levels for RBEU2OS were obtained, and the LET dependency of the RBEU2OS curve was created by linearly complementing the experimental results and scaling the RBEHSG where there were no experimental results. Measurement at more LET levels will be an issue. Second, we recalculated the clinical dose for a single case of osteosarcoma. In future studies, we will analyze the differences due to the beam direction and number of irradiation fields by performing DVH analysis using various cases. Third, we only used U2OS cells and will need to study other tumor cells. However, this study sufficiently demonstrates that dose distribution can vary when recalculated to a tumor-specific dose distribution.

Conclusion

We calculated the dose distribution using RBEU2OS and observed the occurrence of dose distribution degradation, especially in cases with worsening HI. Tumor-specific dose distribution may be non-uniform and impact clinical outcomes.

Acknowledgements

The Authors would like to thank their colleagues at the Department of Radiation Oncology, Shonan Kamakura General Hospital and the Department of Radiation Oncology, Gunma University.

Footnotes

  • Authors’ Contributions

    Conceptualization: S.S.; methodology: S.S. and M.K.; formal analysis: S.S.; investigation: S.S. and M.K.; resources: S.S.; data curation: S.S. and M.K.; writing–original draft preparation: S.S.; writing–review and editing: S.S., M.K., M.O. and T.O.; visualization: S.S.; supervision: M.O. and T.O.; project administration: T.O.

  • Conflicts of Interest

    Tatsuya Ohno received research funding from Hitachi. All other Authors declare no conflicts of interest in relation to this study.

  • Funding

    This work was supported by the following grant: JSPS KAKENHI Grant Number 20K16751.

  • Received August 21, 2023.
  • Revision received September 20, 2023.
  • Accepted September 21, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Kamada T,
    2. Tsujii H,
    3. Blakely EA,
    4. Debus J,
    5. De Neve W,
    6. Durante M,
    7. Jäkel O,
    8. Mayer R,
    9. Orecchia R,
    10. Pötter R,
    11. Vatnitsky S,
    12. Chu WT
    : Carbon ion radiotherapy in Japan: an assessment of 20 years of clinical experience. Lancet Oncol 16(2): e93-e100, 2015. DOI: 10.1016/S1470-2045(14)70412-7
    OpenUrlCrossRefPubMed
    1. Shibuya K,
    2. Ohno T,
    3. Katoh H,
    4. Okamoto M,
    5. Shiba S,
    6. Koyama Y,
    7. Kakizaki S,
    8. Shirabe K,
    9. Nakano T
    : A feasibility study of high-dose hypofractionated carbon ion radiation therapy using four fractions for localized hepatocellular carcinoma measuring 3 cm or larger. Radiother Oncol 132: 230-235, 2019. DOI: 10.1016/j.radonc.2018.10.009
    OpenUrlCrossRef
  3. ↵
    1. Mohamad O,
    2. Imai R,
    3. Kamada T,
    4. Nitta Y,
    5. Araki N, Working Group for Bone and Soft Tissue Sarcoma
    : Carbon ion radiotherapy for inoperable pediatric osteosarcoma. Oncotarget 9(33): 22976-22985, 2018. DOI: 10.18632/oncotarget.25165
    OpenUrlCrossRefPubMed
    1. Koto M,
    2. Demizu Y,
    3. Saitoh JI,
    4. Suefuji H,
    5. Tsuji H,
    6. Okimoto T,
    7. Ohno T,
    8. Shioyama Y,
    9. Takagi R,
    10. Nemoto K,
    11. Nakano T,
    12. Kamada T, Japan Carbon-Ion Radiation Oncology Study Group
    : Multicenter study of carbon-ion radiation therapy for mucosal melanoma of the head and neck: Subanalysis of the Japan Carbon-Ion Radiation Oncology Study Group (J-CROS) study (1402 HN). Int J Radiat Oncol Biol Phys 97(5): 1054-1060, 2017. DOI: 10.1016/j.ijrobp.2016.12.028
    OpenUrlCrossRefPubMed
  5. ↵
    1. Shinoto M,
    2. Yamada S,
    3. Okamoto M,
    4. Shioyama Y,
    5. Ohno T,
    6. Nakano T,
    7. Nemoto K,
    8. Isozaki Y,
    9. Kawashiro S,
    10. Tsuji H,
    11. Kamada T
    : Carbon-ion radiotherapy for locally recurrent rectal cancer: Japan Carbon-ion Radiation Oncology Study Group (J-CROS) Study 1404 Rectum. Radiother Oncol 132: 236-240, 2019. DOI: 10.1016/j.radonc.2018.10.007
    OpenUrlCrossRefPubMed
  6. ↵
    1. Shiba S,
    2. Okamoto M,
    3. Kiyohara H,
    4. Okazaki S,
    5. Kaminuma T,
    6. Shibuya K,
    7. Kohama I,
    8. Saito K,
    9. Yanagawa T,
    10. Chikuda H,
    11. Nakano T,
    12. Ohno T
    : Impact of carbon ion radiotherapy on inoperable bone sarcoma. Cancers (Basel) 13(5): 1099, 2021. DOI: 10.3390/cancers13051099
    OpenUrlCrossRefPubMed
  7. ↵
    1. Inaniwa T,
    2. Kanematsu N,
    3. Matsufuji N,
    4. Kanai T,
    5. Shirai T,
    6. Noda K,
    7. Tsuji H,
    8. Kamada T,
    9. Tsujii H
    : Reformulation of a clinical-dose system for carbon-ion radiotherapy treatment planning at the National Institute of Radiological Sciences, Japan. Phys Med Biol 60(8): 3271-3286, 2015. DOI: 10.1088/0031-9155/60/8/3271
    OpenUrlCrossRefPubMed
  8. ↵
    1. Kanai T,
    2. Endo M,
    3. Minohara S,
    4. Miyahara N,
    5. Koyama-Ito H,
    6. Tomura H,
    7. Matsufuji N,
    8. Futami Y,
    9. Fukumura A,
    10. Hiraoka T,
    11. Furusawa Y,
    12. Ando K,
    13. Suzuki M,
    14. Soga F,
    15. Kawachi K
    : Biophysical characteristics of HIMAC clinical irradiation system for heavy-ion radiation therapy. Int J Radiat Oncol Biol Phys 44(1): 201-210, 1999. DOI: 10.1016/s0360-3016(98)00544-6
    OpenUrlCrossRefPubMed
  9. ↵
    1. Ohno T,
    2. Kanai T,
    3. Yamada S,
    4. Yusa K,
    5. Tashiro M,
    6. Shimada H,
    7. Torikai K,
    8. Yoshida Y,
    9. Kitada Y,
    10. Katoh H,
    11. Ishii T,
    12. Nakano T
    : Carbon ion radiotherapy at the Gunma university heavy ion medical center: New facility set-up. Cancers (Basel) 3(4): 4046-4060, 2011. DOI: 10.3390/cancers3044046
    OpenUrlCrossRefPubMed
  10. ↵
    1. Oike T,
    2. Ogiwara H,
    3. Torikai K,
    4. Nakano T,
    5. Yokota J,
    6. Kohno T
    : Garcinol, a histone acetyltransferase inhibitor, radiosensitizes cancer cells by inhibiting non-homologous end joining. Int J Radiat Oncol Biol Phys 84(3): 815-821, 2012. DOI: 10.1016/j.ijrobp.2012.01.017
    OpenUrlCrossRefPubMed
  11. ↵
    1. Kamada T,
    2. Tsujii H,
    3. Tsuji H,
    4. Yanagi T,
    5. Mizoe JE,
    6. Miyamoto T,
    7. Kato H,
    8. Yamada S,
    9. Morita S,
    10. Yoshikawa K,
    11. Kandatsu S,
    12. Tateishi A, Working Group for the Bone and Soft Tissue Sarcomas
    : Efficacy and safety of carbon ion radiotherapy in bone and soft tissue sarcomas. J Clin Oncol 20(22): 4466-4471, 2002. DOI: 10.1200/JCO.2002.10.050
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Kato S,
    2. Ohno T,
    3. Tsujii H,
    4. Nakano T,
    5. Mizoe JE,
    6. Kamada T,
    7. Miyamoto T,
    8. Tsuji H,
    9. Kato H,
    10. Yamada S,
    11. Kandatsu S,
    12. Yoshikawa K,
    13. Ezawa H,
    14. Suzuki M, Working Group of the Gynecological Tumor
    : Dose escalation study of carbon ion radiotherapy for locally advanced carcinoma of the uterine cervix. Int J Radiat Oncol Biol Phys 65(2): 388-397, 2006. DOI: 10.1016/j.ijrobp.2005.12.050
    OpenUrlCrossRefPubMed
  13. ↵
    1. Imai R,
    2. Kamada T,
    3. Araki N, Working Group for Carbon Ion Radiotherapy for Bone and Soft Tissue Sarcomas
    : Carbon ion radiotherapy for unresectable localized axial soft tissue sarcoma. Cancer Med 7(9): 4308-4314, 2018. DOI: 10.1002/cam4.1679
    OpenUrlCrossRefPubMed
  14. ↵
    1. Fowler JF
    : The linear-quadratic formula and progress in fractionated radiotherapy. Br J Radiol 62(740): 679-694, 1989. DOI: 10.1259/0007-1285-62-740-679
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Schlaff CD,
    2. Krauze A,
    3. Belard A,
    4. O’Connell JJ,
    5. Camphausen KA
    : Bringing the heavy: carbon ion therapy in the radiobiological and clinical context. Radiat Oncol 9(1): 88, 2014. DOI: 10.1186/1748-717X-9-88
    OpenUrlCrossRefPubMed
  16. ↵
    1. Wang JZ,
    2. Huang Z,
    3. Lo SS,
    4. Yuh WTC,
    5. Mayr NA
    : A generalized linear-quadratic model for radiosurgery, stereotactic body radiation therapy, and high-dose rate brachytherapy. Sci Transl Med 2(39): 39ra48, 2010. DOI: 10.1126/scitranslmed.3000864
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Permata TBM,
    2. Sato H,
    3. Gu W,
    4. Kakoti S,
    5. Uchihara Y,
    6. Yoshimatsu Y,
    7. Sato I,
    8. Kato R,
    9. Yamauchi M,
    10. Suzuki K,
    11. Oike T,
    12. Tsushima Y,
    13. Gondhowiardjo S,
    14. Ohno T,
    15. Yasuhara T,
    16. Shibata A
    : High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells. J Radiat Res 62(5): 773-781, 2021. DOI: 10.1093/jrr/rrab050
    OpenUrlCrossRef
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Dose Distribution Degradation of Carbon-ion Radiotherapy Caused by Tumor Cell-specific Relative Biological Effectiveness of Osteosarcoma: A Simulation Study Using In Vitro Experimental Results
SHINTARO SHIBA, MOTOHIRO KAWASHIMA, MASAHIKO OKAMOTO, TATSUYA OHNO
Anticancer Research Nov 2023, 43 (11) 4873-4878; DOI: 10.21873/anticanres.16684
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords