Therapeutic irradiation in the management of gynecological cancer and predictability of radiation-induced complications

Терапевтичне опромінення хворих онкогінекологічного профілю та можливість прогнозу променевих ускладнень Іванкова В. С.1, ORCID: 0000-0001-5750-6852, e-mail: valentina_ivankova@ukr.net Михайленко В. М.2, ORCID: 0000-0003-4922-6214, e-mail: mvmik@yahoo.com Дьоміна Е. А.2, ORCID: ORCID: 0000-0002-1058-0489, e-mail: edjomina@ukr.net Хруленко Т. В.1, ORCID: 0000-0002-2468-3390, e-mail: khrulenko@ukr.net Барановська Л. М.1, ORCID: 0000-000-1018-6377, e-mail: lidabaranovska@ukr.net Грінченко О. О.2, ORCID: 0000-0002-9865-4453, e-mail: griniolia@gmail.com 1Національний інститут раку Міністерства охорони здоров’я України, Київ, Україна; 2Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького Національної академії наук України, Київ, Україна

and peptides in gynecological cancer patients and healthy donors to predict the risk of radiation-induced complications. Results and discussion. Given the delayed effect of RT, the treatment effectiveness was analyzed immediately after RT session and also 3 months upon completion of the conservative therapy. Thus, positive tumor response upon three months of observation over time was registered in 67.9 ± 5.2% of patients in study group I, in 72.5 ± 6.9% in study group II, and in 51.3 ± 6.8% in comparison group. Hence the values in study groups were higher than in comparison group by 16.6% and 21.2% respectively. All patients tolerated BT satisfactorily. Neither general nor local rectum or bladder severe (above grade II) toxicities were noted both during treatment and over the next 3 months upon its completion in all the patients regardless of study group. Results obtained in radiobiological studies correlated with clinical and literature data. Conclusions. Decrease in manifestations of RT toxicity, namely of the early radiation reactions from interfacing critical organs was established in groups I and II vs comparison group I, especially in group I where a highenergy 192 Ir source was used in the SBD irradiation mode of 3 Gy twice a week. The obtained results of the experimental study suggest that the content of SH-groups in blood plasma and the level of PBL apoptosis can be considered as additional predictive measures of radiosensitivity of non-malignant cells from the irradiated tumor environment.

Relationship with academic programs, plans and themes
The study was performed in the framework of the planned research project at Research Department of Radiation Oncology of National Cancer Institute of the Ministry of Health of Ukraine "To determine the optimal methods of high-energy brachytherapy with a source of ionizing radiation of iridium-192 in the treatment of patients with secondary vaginal cancer tumors considering the molecular biology of tumor" (State Registration

INTRODUCTION
Today, applying radiotherapy (RT) in the management of both primary and secondary vaginal cancer (SVC) take pride of place in the spectrum of specialized treatments for cancer patients. Indications for the surgical treatment in such cases emerge seldom. Specifically, an extended hysterectomy with removal of a part of vagina is effective in the young and middle-aged women with localization of tumor in the upper third of vagina. Surgical extirpation is often not possible, since to perform a radical surgery the exenteration is required because of proximity of bladder and rectum. Therefore, RT is a choice in about 80% of cases of primary vaginal cancer and even more when dealing with SVC [1][2][3][4][5][6].
At present, the national clinics are built up with advanced equipment for the contact RT by means of a remote afterloading method by the high-dose rate sources (HDR-therapy). The 192 Ir sources of high dose-rate gamma radiation on the HDR device "Gamma Med plus IX" (Germany) are continuously used at Radiation Oncology Department of the National Cancer Institute. This installation is a new generation device for contact RT, which enables intracavitary radiation therapy (IRT) by means of the remote afterloading method in a combined radiation treatment of SVC patients and patients with other localization of cancer. This technology of a remote automatic loading of the ionizing radiation source (IRS) after the applicator placement makes it possible to adjust the dosimetric calculation before the source placement and start of irradiation. There is one 192 Ir source in the device, and the former moves along the centering catheter in steps of 2.5 or 5 mm. The maximum number of active positions in the centering catheter is 48, which allows to widely change the size of irradiated target (up to 12 cm long) with further dosimetric planning and optimization of dose distribution under fractional irradiation. Ability to deliver the maximum doses of radiation directly to the tumor with minimal exposure to critical and related organs is an important advantage of IRT (brachytherapy -BT). The SVC treatment protocol in addition to BT includes the external-beam radiotherapy (EBRT) (syn. teleradiotherapy). Combination of these two methods is a "gold standard" for the treatment of tumors of the female reproductive system worldwide. Over the last decade it has emerged as a high-tech, effective, organ-preserving, and state-of-the-art component of radical radiation treatment of gynecological cancer, enabling a significant reduction of radiation exposure to the critical and interfacing organs [14,[16][17][18][19]. Chemotherapy is used only as a component of comprehensive treatment and as a palliative antineoplastic tool if RT is unavailable.
From 14% to 80% of gynecological cancer patients receiving RT can as a result experience a significant number of local radiation reactions of varying intensity, which worsen the quality of life, lead to forced breaks in treatment, and affect its results [20].
The issue dealing with affecting normal cells that fall under the area of therapeutic irradiation of cancer patients draws attention not only of radiation oncologists, but also clinical radiobiologists. Despite the conformal strategy of radiation therapy, some normal cells from the tumor environment are inevitably exposed to radiation, which can lead to the development of distant radiation-induced complications, including secondary cancer [7,12,[21][22][23][24][25]. Molecular, chromosomal and other abnormalities in healthy cells of primary cancer patients change the functional status of these cells, including radiosensitivity, and consequently afford ground to consider them to be only conditionally normal [26][27][28][29][30]. Additional radiation-induced ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
The sulfhydryl (SH-) groups of proteins and low molecular weight compounds can be appropriately attributed to such biomarkers. They are of a key role in the numerous biological processes, particularly in apoptosis, proliferation, metabolism and regulation of transcription. The exchange of thiol-disulfide has been found to play an important role in protein folding of its stability [37,38] as well to affect the redox potential of cells and proteins [39]. The active forms of oxygen and nitrogen (AFON) being essential in these processes are produced both in normal physiological pathways of cellular function and in response to harmful factors, including radiation, and precede the formation of cellular genetic instability. Herewith a correlation between the apoptosis-associated changes in cells and their radiosensitivity was substantiated [40][41][42]. The increased induction of chromosomal aberrations and formation of micronuclei in lymphocytes of cancer patients after irradiation in vitro by contrast with healthy donors, which was observed in a number of studies [43,44], may be explained in many cases by apoptosis as a mechanism of elimination of cells with damaged DNA and maintaining the genetic stability.
Purpose -to enhance the effectiveness and assess the toxicity of SVC RT via ascertaining BT (brachytherapy) most suitable techniques depending on the type of ionizing radiation and exploring predictability of radiation-induced complications in terms of biomolecular cell properties.

MATERIALS AND METHODS
There are appropriate guidelines for SVC, which mainly take into account the tumor size and method of primary RT. There are no fundamental differences between EBRT and BT in the mechanism of radiation effect, which leads to cell death, damage to normal tissues and tumor destruction. Radiobiological basis of both RT variants have a lot in common, however, their physical features of spatial and temporal dose distribution differ significantly. The main provisions defining BT effectiveness are the dose-rate value of contact radia-ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
Під час проведення поєднаної променевої терапії (ППТ) з використанням радіоактивного 192 Ir враховувались показання та протипоказання до внутрішньопорожнинної гамма-терапії. В ході лікування tion with low dose rate (LDR) and the model of fractional exposure when using the HDR sources. Irradiation with a medium dose-rate source is referred to as MDR. When applying LDR irradiation the time of RT session is significantly extended. Administration of the fractionated BT with a high dose-rate leads to greater damage to normal tissue cells compared to the tumor ones. Prolonging the RT session reduces the incidence of early radiation reactions, but worsens the results of local treatment, with no effect on the number of late radiationinduced complications [13,16]. Choice of IRS, total focal doses (TFD), and fractionation mode are determined by the spread of process, equipment of the healthcare institution and preceding EBRT. Methods of vaginal tumor RT are highly variable, which greatly complicates t he choice of the most optimal option for each patient [14,[16][17][18][19].
Effectiveness of the new 192 Ir radiation source application in contact PT was studied at the National Cancer Institute Clinic (Radiation Oncology Department) using a device for high-energy BT with a HDR gamma radiation source. Patients with SVC (n = 106) previously treated for CC (n = 65) or UC (n = 41) were enrolled. Patients with locally advanced pelvic tumor stage II-III, T2-3N0-1M0 were prevalent in the study group.
Clinical diagnosis in all patients was confirmed by the histological study. In most cases of secondary cancer upon primary CC the squamous cell carcinoma was found, while the adenocarcinoma was mostly diagnosed after primary UC.
In order to determine the boundaries of tumor process, its features, state of critical organs, and presence or absence of concomitant diseases all the patients had undergone a comprehensive examination. The latter included clinical check-up to determine visual and palpatory parameters of primary tumor, comprehensive ultrasonographic examination, computed tomography (CT) of the chest, abdomen and pelvis, magnetic resonance imaging (MRI) of the pelvis, cystoscopy and fibrorectosigmoscopy (assay of spread and presence or absence of lesions that may complicate the chemoradiotherapy (CRT) and/or lead to undesirable toxic complications and thus necessi-tate the dose reduction), and laboratory tests.
Indications and contraindications for the intracavitary gamma therapy were taken into account when performing combined radiation therapy (CRT) using a 192 Ir source. EBRT was administered during the treatment of studied patients. The radiation dose was planned taking into account the previously performed radiotherapy.
Upon comprehensive examination all the patients had received CRT, namely conformal EBRT on the linear electron accelerator device and contact RT.
of RT using a HDR device "GammaMed" with 192 Ir radiation sources. BT was conducted in the main group I (n = 37) according to the irradiation regime of SFD 3 Gy twice a week up to the TFD of 30 Gy in 10 fractions. The main group II (n = 34) had received the irradiation regimen of 6 Gy SFD 1 time per week up to 30 Gy TFD in 5 fractions. The MDR RT from 60 Co was administered to the comparison group (n = 35) upon EBRT. BT was delivered according to the developed protocol via irradiaion regime of 6 Gy SOD twice a week up to 40-42 Gy TFD. The gamma-ray teletherapy was accordingly admi-nistered to the injury zone of regional metastases. At the same time, the doses of previous RT were necessarily taken into consideration.
CRT was provided to all study subjects along with administering chemoradiomodifying agents, namely tegafur orally 235 mg/m 2 BID throughout the course of CRT and cisplatin 30 mg/m 2 intravenously once a week for 4-6 weeks up to the 200-300 mg total course dose (TCD).
Statistical processing of the obtained clinical findings included calculation of primary statistical parameters, identification of intergroup differences by the statistical criteria, establishing the relationship between variables using parametric and non-parametric correlation analysis by means of the Wald-Wolfowitz test [45].
Along with clinical study, the radiobiological research was conducted to count the apoptotic cells in both intact and irradiated peripheral blood lymphocytes (PBL), as well as the level of SH-groups of plasma proteins and peptides in gynecological cancer patients and nominally healthy individuals.
The study was aimed to ascertain and further predict the nature of radiation disorders developing in tissue cells from the tumor environment, including PBL, which are also exposed to radiation [15,46].
Decision was taken to carry out the study applying metformin (MET) radiomodifier ex vivo, which, according to the literature, increases the sensitivity of tumor cells to radiation [47]. The authors suggest that its antitumor effect is associated with inhibition of the 1st respiratory complex of oxidative mitochondrial phosphorylation. The study [48] has made clear that MET directly increases antitumor cytotoxicity of blood lymphocytes.
Відгук на проведену ППТ безпосередньо після завершення повного курсу лікування (сумарний пози-added to the blood as a solution in PBS (1:20) to achieve a final concentration of 2 mM and 20 mM in the blood samples. Irradiated blood samples were carefully (without active stirring) transported for 10-12 minutes at 4 o С in an opaque thermostated container for biological samples; after that plasma and lymphocytes were immediately isolated from them for further studies. Blood plasma was obtained by centrifugation (15 min at 1200 g; 20 o C) and stored at 20°C up to 3 days. PBL isolation was achieved using the HISTOPACK-1077 HYBRI-MAX according to the manufacturer's manual (BioReagent) [49]. The content of hypodiploid cells in PBL samples was assessed by flow cytometry (DxFlex, Beckman Coulter Biotechnology Co. Ltd with Kaluza C software for clinical analysis) after preliminary processing the cells by ribonuclease and propidium iodide staining according to the technique described in [50]. Evaluation of the content of SH-groups of proteins and peptides in blood plasma (SH-test) was performed by means of spectrophotometric method in modification [51]. Statistical processing of the results was performed via common methods of variation statistics, correlation and regression analysis. The difference between the obtained values was considered to be significant at p ≤ 0.05 [52].

RESULTS AND DISCUSSION
Effectiveness of the CRT using 192Ir in SVC patients was analyzed taking into account the rate of tumor regression, presence of radiation-induced reactions and complications that occurred both during treatment and upon its completion.
According to the World Health Organization (WHO) GUIDELINES, the tumor regression was determined based on a follow-up of clinical parameters of the tumor process in comparison with data obtained using modern imaging tools (comprehensive sonographic examination, CT, MRI).
Regression of exophytic component and infiltrative changes of primary tumors under the HDR RT occurred, as a rule, by the end of IRT session at the 30 Gy dose level.
The response to CRT immediately upon completion of the full course of treatment (the total positive effect) ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
Всі пацієнтки задовільно переносили ВППТ. У жодної з них, незалежно від групи, упродовж лікування і в найближчі 3 місяці після його завершення, не відмічено тяжких (вище II ступеня) загальних проявів токсичності з боку сечового міхура та прямої кишки. was more pronounced in patients of the study groups. In particular, the analysis of effectiveness of 192 Ir sources in HDR BT and of follow-up data during the CRT course showed that the positive tumor response (complete + partial regression) upon the full course had increased by 12.7% in group I and by 15.9 % in group II compared with the use of 60 Co MDR in BT.
Given the delayed effect of RT, the effectiveness of treatment was also analyzed 3 months upon completion of conservative therapy. Specifically, after a three-month follow-up a positive tumor response was registered in 67.9 ± 5.2% of patients in group I, in 72.5 ± 6.9% of subjects in group II, and in 51.3 ± 6.8% of cases in the comparison group. Hence the values in study groups were higher than in the comparison group for 16.6% and 21.2% respectively. No signs of tumor progression were detected within 6 months of the follow-up in any patient.
Thus, according to the obtained findings of CRT administration in SVC patients, it was found that providing HDR BT via a high-energy 192 Ir source according to the developed techniques, increased the frequency and degree of tumor regression in the main groups I and II vs comparison group receiving the MDR BT using a 60 Co radiation sources.
The toxicity of the performed RT was evaluated according to the RTOG/EORTC (1995) classification [11]. The treatment toxicity in the number and degree of its manifestations in the groups of oncogynecological patients differed a little from the comparison group and did not exceed grade II. As for the general toxicity, there was a slight nausea requiring no medical correction within treatment period in the vast majority of SVC patients in all study groups. Neither severe neutropenia nor thrombocytopenia were observed. The patient's condition returned to normal in about a month after treatment. No manifestations of the late general toxicity were observed in any patient at examination up to 6 months upon treatment.
Radiation reactions of vaginal mucosa attributed to the local reactions were mild or moderate and manifested as hyperemia of mucosa or limited membranous epitheliitis (grade II of toxicity) in the vast majority of patients in all study groups. Membrane epitheliitis of vagina was more often observed in patients with a compromised history, patients with an exophytic component of the tumor in destruction stage, which was accompanied by the contamination with pathogenic flora. It was noted that radiation reactions of grade II were almost absent in the main groups. Sanitation of vagina with antibacterial preparations was administered to the patients during treatment, which allowed continuing the RT course until its completion.
All patients tolerated the IRT satisfactorily. There were no severe (above grade II) general manifestations of toxicity from bladder or rectum during treatment and in the next 3 months upon its completion in any patient, regardless of group.
hypodiploid cells, was determined in lymphocytes of the NHI and ND UC patients. Typical histograms of the fluorescence intensity of PBL are presented in Fig. 1.
The PBL apoptosis level in healthy donors was 1.91 ± 0.38%. The mean group level of PBL apoptosis under TI (therapeutic irradiation) increased linearly and in proportion to radiation dose, exceeding the value of intact control from 24% to 52% (р ≤ 0.05) (Fig. 2, A).
The level of PBL apoptosis in the oncogynecological patients was 3.50 ± 0.29%, being 1.8 (р ≤ 0.05) times higher than in the NHI. However, under the influence of TI the average group level of apoptosis of PBL in patients, in contrast to NHI, almost did not change with dose increase (Fig. 2, B).
Кількість SH-білків і пептидів визначали спектрофотометрично в інтактній плазмі крові як УЗО, так application of MF in different doses, the lower of which was close to therapeutic dose, and the larger one was in a range used in experiments on cell cultures. Incubation of the NHI blood samples with MF at a concentration of 2 mM and 20 mM significantly increased (2-fold and 1.7-fold respectively, р ≤ 0,05 ) the percentage of hypodiploid cells (Fig. 2, A).
Another pattern of changes in the apoptosis level was observed in PBL of patients under applying different MF doses. The tendency of apoptosis level to increase by 12% (p ≤ 0.05) was observed when incubating LPC with 2 mm MF, however, when using a higher dose of the drug, the percentage of hypodiploid cells decreased by 30% (p ≤ 0.05) compared with intact LPC patients ( Fig. 2, B).
Quantification of viable cells using the trypan blue supravital staining showed no significant difference between the intact PBL and ones incubated with MF. The comparison of data on the overall PBL survival and the level of apoptosis in them indirectly indicates the MF effect on inhibition of mitochondrial respiratory processes and cellular gluconeogenesis, namely the inhibition of complex I of mitochondrial respiratory chain [53]. Consequently, the oxidative phosphorylation was suppressed and ATP content was reduced, being, in its turn, one of the proapototic signals.
Under a joint effect of MF and TI in different doses, the number of apoptotic cells in PBL of NHI increased similarly to the changes registered in comparison group (Fig. 2, A). Thus, the percentage of hypodiploid cells increased linearly and in proportion to the radiation dose, exceeding the value of non-irradiated control for 12% to 48% (р ≤ 0.05). Incubation of blood with added MF in different doses and subsequent TI was not accompanied by an increase in PBL apoptosis level, as well as in the cells that were irradiated with no MF addition (Fig. 2, B). In contrast, there was a tendency to decrease the percentage of hypodiploid cells in groups incubated with MF in different doses.
Thus, a linear increase in the PBL percentage in the state of apoptosis was registered upon joint effect of MF and TI in vitro on blood samples from NHI, which indicates a significant sensitization of cells to IR under the action of MF. The obtained results correlate with literature data, where MF exhibited the radioprotective and radiosensitizing properties in normal and tumor cells [54]. However, no described effects have been observed in the studied patients, which may indicate changes in mitochondrial respiratory processes and cellular gluconeogenesis associated with malignant process.
Studying the content of total SH-groups in blood plasma were performed considering their key role in regulation of redox balance both in normal conditions and under the enhanced AFON production, which is observed under the impact of IR and/or presence of a pathological process.
The amount of SH-proteins and peptides was determined spectrophotometrically in the intact plasma of both NHI and patients by the content of colored 2-nitro-5thiobenzoate anion with a maximum absorption at 412 nm.
The content of sulfhydryl groups of proteins and peptides in the blood plasma of oncogynecological patients was reduced in comparison with NHI (Fig. 3). It was found that the average value of content of sulfhydryl groups in the blood plasma of patients was 0.42 ± 0.024 mM, which was by 18% less than in the NHI group (0.51 ± 0.014 mM). When irradiating blood samples from patients, there was a slight increase (up to 4%) in the number of SH-groups compared to their value in samples without TI, correlating with data in NHI, where the increase in SH-groups was 6% (Fig. 3).
Thuswise, insignificant changes in content of SH-groups in plasma of the NHI under TI in a dose range of 0.5-3.0 Gy are explained by manifestation of compensatory capabilities of the antioxidant system, which neutralizes the AFON excess formed by X-ray exposure. The blood plasma of patients contained a lower number of SH-groups in comparison with NHI, indicating the redox balance change towards oxidative processes. Incubation of blood ex vivo with added MF preparation to the plasma did not affect the level of SH-groups.

CONCLUSIONS
1. The study has made it possible to ascertain decreased RT toxicity manifestations, i.e. early radiation reactions from tumor-adjacent critical organs in groups I and II compared with comparison group, especially in group I, where a high-energy 192 Ir radiation source was used at SFD = 3 Gy twice a week. Applying lower single doses of radiation has led to a decreased incidence of early radiation reactions with a little effect on the number of late radiation complications, which is important in predicting them during treatment. Decrease in percentage and grade of early local radiation reactions on vaginal mucosa, as well as their number and grade in critical organs when providing 192 Ir HDR BT is most likely due to a sharp контроль узо контроль онкогінекологічних хворих ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
3. Одержані результати свідчать, що вміст SH-груп у плазмі крові та рівень апоптозу ЛПК можна вважати додатковими прогностичними показниками радіочутливості немалігнізованих клітин із оточення опромінюваної пухлини, що можна враховувати при прогнозуванні променевих ускладнень здорових тканин. 2. The study shows that oncogynecological patients, in contrast to NHI, are characterized by a significant (1.8-fold) increase in the level of spontaneous apoptosis in lymphocytes and a decreased (by 18%) content of SH-groups in peripheral blood plasma. The testing X-ray irradiation of blood in vitro in the dose range of 0.5-3.0 Gy caused a dose-proportional increase by 24%-56% of apoptotic death of PBL of relatively healthy donors, but did not affect the level of apoptosis of PBL from patients. Simultaneously, a slight dose-dependent increase in the number of SH-groups in the blood plasma of patients as well as NHI (4% and 6%, respectively) was registered. Peculiarities of the antidiabetic drug MF effect on the level of hypodiploid cells in lymphocyte population and the content of SH-groups in the blood plasma of cancer patients in comparison with NHI were determined. The protective effect of MF, when having the patients' blood exposed to TI, manifested in the form of maintaining the normal concentration of SH-groups in plasma and the level of apoptosis in PBL in contrast to NHI, where the level of apoptosis increased in proportion to the radiation dose.