Morphological substantiation of radiomodification of preoperative radiotherapy in patients with advanced endometrial cancer

Relevance. Endometrial cancer ranks first among oncogynecological diseases, however, 5-year survival can be achieved only in 67,7 % of patients, while 22,4 % of patients die during this period from the con­tinuation of the tumor process. Therefore, improving the complex therapy of patients with EC is very important. 
Purpose of the study. to investigate therapeutic pathomorphosis in pa­tients with advanced endometrial cancer with neoadjuvantchemoradiation therapy. 
Materials and methods. In order to study radiation pathomorphosis, 26 patients with ER T1-3N0-1M0-1 were examined. Group I consisted of 11 patients with a preoperative course of external radiation therapy SOD 30 Gywithout radiomodification, II – 15 patients with a preoperative course of external radiation therapy SOD 30 Gywith radiomodification. 
Results and its discussion. When conducting a comparative assessment of radiation pathomorphism in patients of the study groups, it was found that when using the preoperative course of TFD on Gy 30 with radiomodi­fication, a significant decrease in the mitotic index occurs. There is also a tendency to an increase in the degree of tumor regression, the number of pathological mitoses and stromal reactions during the preoperative course of TFD on 30 Gywith radiomodification. 
Conclusions. It was found that radiation therapy with EС with radiomodi­fication gives a more positive effect and regression of more than 50,0 % of the tumor volume, a decrease in its biological potential. The volume of the damaged tumor was 58,0 %, and the volume of the residual tumor was 42,0 %, that is, most of the tumor mass underwent regression.


INTRODUCTION
Over the past two decades, Ukraine has experienced an increase in the incidence of endometrial cancer (EС), which, according to the Bulletin of the National Chancellor of the Register of Ukraine, currently accounts for 28 cases per 100,000 of the female population (26.4 in 2007) while the mortality rate indicator reached 6.8 per 100,000 female in 2008 [1,2].
Evaluation EC treatment outcomes indicates a high efficiency of combination therapy at the early stages of the disease, which allows achieving five-year survival in 85-95 % of patients with stage I of the process, in 65-70 % with stage II and only 29 % with stage III. Thus, the combined treatment method can be effective in treatment of only 70-75 % of patients with EC with operable tumors without somatic contraindications to surgical treatment [5].
The development of EC relapses and metastases is most often caused by dissemination and implantation of tumor cells (ТC) in the area of the surgical field, lymphogenous and hematogenous spread of them during surgery, as well as the relative radioresistance of endometrial adenocarcinoma. The analysis of the frequency of development of EC relapses and metastases depending on location showed that in patients undergoing combined treatment, relapses were more frequently observed on the vaginal scar (6,4 %), metastases were more often detected in the vagina (2,7 %), in the peripheral lymph nodes (3,2 %), in the abdominal cavity (4,8 %) and lungs (3,7 %) [6].
In this regard, according to some authors, one of the most effective methods of increasing efficiency in patients with common EС is preoperative radiation therapy, which contributes to a significant decrease in the biological potency of ТС to metastasize and to decrease their ability to implant, which reduces the risk of local and general dissemination during further surgery. Proponents of this approach believe that operability is improved and blockade of the lymphatic system is achieved, which enhances the long-term results of treatment by 10-20 % [7,8].
The ranges of doses used in preoperative irradiation vary from 30 to 60 Gyfor prolonged courses and from 20 to 30 Gy for intensely concentrated courses. However, the completeness of the local effect is a prerequisite for permanent cure and increased survival after irradiation while the frequency of local or regional relapses correlates with the total dose and also depends on the amount of exposure. Morphological changes in the tumor are also one of the criteria of the effectiveness of preoperative radiation therapy. At the same time, a high frequency of reactions and complications from normal organs and tissues prevents increasing therapeutic dose and the amount of radiation exposure. To increase the frequency and persistence of local cure along with maximally preserving critical organs and tissues, increasing survival, further improvement of radiation treatment methods is necessary [9].
The aim of the study was to investigate therapeutic pathomorphism in patients with advanced endometrial cancer with neoadjuvant chemotherapy and radiation therapy.
The preoperative course of remote radiation therapy was carried out using ROKUS-AM or Clinac-600 apparatus via classical fractionation mode into the pelvic area and regional metastasis pathway, the total focal dose of TFD at A/B points of 30 Gr. Irradiation was performed (5 times a week), a single focal dose (SFD) of 2 Gr. In order to radiomodify the preoperative course of distant radiation therapy, patients with EC of the study group were administered a solution of cisplatin 100 mg once a week.
Surgical treatment was performed in the amount of expanded hysterectomy with appendages and omentum resection.
In all examined patients, the diagnosis was verified morphologically before surgery. The morphological study took into account the nature of the tumor growth (exophytic, endophytic, mixed), histological structure, tumor differentiation degree, depth of tumor invasion into the myometrium, mitotic activity, the number of pathological mitoses, nature of cellular reactions in the tumor stroma, presence and severity of dystrophic and regressive changes in the tumor, as well as presence of necrosis, apoptosis.
Surgical material for morphological studies was taken from the central and peripheral areas and preparations were prepared according to standard methods. Histological preparations were stained with hematoxylin and eosin and according to the Van Gieson method. An ocular ruler was used to determine the volume of the residual tumor [55].
The mitotic index and the number of pathological mitoses were determined by the method of I. A. Alova and A. I. Kazantseva, radiation pathomorphism, the degree of radiation damage to the tumor, and the nature of cellular stromal reactions were determined by the method of T. P. Yakimova [60].
The degree of radiation pathomorphism was assessed in accordance with a 4-point scale: Degree I of radiation damage was taken as 1 relative unit. It corresponded to minor changes in the structure of the tumor and regression of single complexes.
Degree II of radiation damage was determined in those cases when there was focal disappearance of the parenchyma, appearance of necrosis and uneven increase in the volume of the stroma. They were given a rating of 2 relative units.
Degree III of radiation damage was assessed in those cases when the volume of the stroma prevailed over the volume of the tumor with focal preservation of the tumor areas, which was estimated by 3 relative units.
Degree IV of radiation damage corresponded to complete tumor regression and was evaluated by 4 relative units.
При обробці зон регіонарного метастазування першу третину курсу променеве лікування проводили зустрічними полями розмірами 16 × 16 см dystrophic changes in tumor cells of degree 1 was evaluated as 1 relative unit in cases where the dystrophic changes were insignificant and were characterized by a slight increase in the size of cells and nuclei, condensation of chromatin with its placement along the edge of the nucleus, weak hypertrophy of the nuclei slight vacuolization of the cytoplasm, moderate heterochromia.
Moderately expressed dystrophy of tumor cells was evaluated by 2 relative units in the case when a clear polymorphism of cells developed, increased volume of cells and nuclei was observed, fragmentation of nuclei and the appearance of multicore, gigantism of cells and nuclei, pycnosis of the latter, fuzzy between cells and nuclei, cell lysis, also destroyed cell groups, incompletion of individual tumor structures were detected.
The pronounced degeneration of tumor cells was determined during vacuolization and nuclei lysis, pronounced fatty degeneration of the cytoplasm, with a sharp polymorphism and destruction of complexes with the formation of cribriform structures and was evaluated by 3 relative units.
Stromal cell responses of the tumor bed were also evaluated by a 3-point scale depending on the morphological state of the connective tissue, given the condition of the fibers and the main substance of the connective tissue. In the presence of sclerotic changes, a small number of fibrocytes and the absence of fibroblasts, cellular stromal reactions were evaluated by 1 relative unit. While maintaining almost normal structure of connective tissue, fibrous structures, and fibroblast cell elements, the state of the stromal cell reaction was evaluated by 2 relative units. With a pronounced local immune cell response, the nature of stromal changes corresponded to 3 relative units [48]. The apoptosis index was calculated by the number of apoptotic cells per 100 tumor cells.
Treatment of patients with EС was surgical, combined and complex depending on the stage of the disease and the histological structure of the tumor. Indications for surgical intervention were: EС T1a-3N0-1M0-1; general satisfactory condition of the patient; a combination of EC with uterine fibroids or tumors of the uterus; chronic inflammation of the appendages with frequent exacerbations; pyometra; the risk of perforation due to the tumor invading all layers of the uterus. After laparotomy, abdominal, pelvic, and paraaortic lymph nodes were examined.
The collection of material for a cytological study in order to clarify the stage of the disease and to study the ablastics of surgical interventions was taken from the Douglas space immediately after laparotomy, from the site of the surgical field after removal of the uterus and from the surface of the vaginal scar at the end of the surgery.
The removed preparation was subjected to macroscopic examination externally and in section. In this case, the size of the uterus, the thickness of the myometrium, the location and form of tumor growth, the depth of invasion of the myometrium were evaluated.
cm or 18 × 18 cm in the iliac and sacral-gluteal areas up to TFD 15 Gy. Then, with static irradiation, four opposite fields were used (two on the iliac and two on the sacro-gluteal size 6 × 16 cm, in pairs, parallel to each other, with a distance between the medial limits of 2 cm). EC T2-3N0-1M0 patients of both groups were additionally irradiated in a vaginal scar region using an AGAT-B apparatus by adding an ovoid system to it. A single focal dose was 35 Gy, TFD 28 Gy for 8 fractions.
A morphological study made it possible to find that more often in patients of both groups there was a moderately differentiated endometrial adenocarcinoma: 45.5 and 46.7 %. In general, adenocarcinoma was detected in almost 90 % of patients.
Indicators of radiation pathomorphosis in EC patients of Group 1 are shown in Table 3.
Evidently, the degree of radiation damage to the tumor at the tissue level was moderately pronounced. The tumor mass, which succumbed to regression, was replaced by newly formed connective tissue.
The degree of dystrophic changes in ТС was also moderately pronounced. In one case (9.1 %), complete (IV degree) tumor regression was observed, while in other 2 cases (18.2 %), a significant degree of radiation damage to the tumor when separate foci of ТC remained in its place, which amounted to 12-13 % of the tumor masses, was observed.
Thus, remote radiation therapy of endometrial cancer TFD 30 Gy leads to significant radiation damage at the tissue level and full regression of 27.3 % of the tumor. Most malignant neoplasms (76.9 %) are susceptible to fatty degeneration (Fig. 1).
Additionally it is expressed in increased TC dimensions in certain cases, partially in cytolysis and karyolysis. Two patients (18.2 %) showed significant necrosis in the tumors. In one case it was a lowdifferentiated adenocarcinoma, and in another one, adenocarcinoma of moderate differentiation degree was observed. It should be noted that the degree of radiation damage to the tumor at the tissue level, i.e. tumor regression, did not correlate with necrosis presence. While in all observation cases characterized by necrosis in tumors, there was a pronounced dystrophy of the TC.
Regarding apoptosis index, its average value is very low, i.e. 2.96 ± 0.94, which is still 1.8 times higher than this value in intact tumors, 1.46 ± 0.33. Therefore, the role of apoptosis in the regression of endometrial tumors remains insignificant.
The nature of stromal cellular and immune reactions in EC was quite active and according to a 3-point scale it has 2.53 ± 0.96 relative units. Upon that, in 54.5 % of tumors, (6 out of 11) infiltration of the tumor and surrounding tissues by immunocompetent cells, lymphocytes predominantly, was revealed. Thuswise, half of EC tumors have a cellular nature of stroma reactions.
Radiation pathomorphosis of EC in patients of Group I depending on the form of tumor growth is shown in Table 4.
The analysis of the study outcomes (Table 4) shows that the greatest degree of radiation damage of the tumor at the tissue level is observed when a tumor is localized within the mucous membrane. This is quite clear, since a tumor, being under radiation therapy influence, is not shielded by the surrounding tissues. In addition, it is affected by compensatory-protective and immune reactions of the stroma. With this nature of growth, the highest rate of degenerative changes in TC is observed.
Being characterized by such nature of EC growth, the proliferative properties of the tumor are completely suppressed, and the stroma is densely infiltrated by immunocompetent cells, which have a cytotoxic effect on the tumor. The apoptosis index is moderate.
in combination with direct action of PT, provides a good antitumor effect. In endophytic EC, which is the most common one, the following is observed: moderate degree of radiation damage to the tumor at the tissue level, moderate degree of dystrophic changes in the TC, significant decrease in mitotic activity of tumor tissue and moderate degree of cellular stromal reactions. They are considered to be positive outcomes and they are most likely achieved by both radiation and protective-compensatory reactions in the myometrium. In mixed tumor growth, tumor regression degree is the lowest.
The dependence of EC radiation pathomorphosis in patients of Group I on the histological structure of the tumor is presented in Table 5.
The data provided in Table 5 show, that at the tissue level, a moderate degree of regression occurred in adenocarcinomas with moderate and low degree of differentiation, while pronounced dystrophic changes, like in other groups, were typically occurring in lowgrade differentiated adenocarcinoma and rare forms. At the same time, the proliferative activity of tumors was mostly inhibited in highly and moderately differentiated adenocarcinomas.
It is to be emphasized that the intensity of cellular stromal reactions, including immune ones, is the highest in adenocarcinomas of moderate and low degree of differentiation. And this local immune response is not suppressed by RRT TFD 30 Gy.
Apoptosis study outcomes have showed that the regression of tumors influenced by radiation therapy occurs via dystrophy and necrosis. Moreover, low-grade differentiated tumors mostly tend to be affected. The effectiveness of RRT of endometrial cancer via TFD 30 Gy on our material also depended on the depth of tumor invasion into the myometrium, that is represented in Table 6.
The outcomes provided in table 6 show that the highest degree of radiation damage was observed in tumors with invasion of the myometrium of 0.5 cm and less. These data correlate with the results of the study of radiation pathomorphosisof tumors localized within the mucous membrane and characterized by exophytic form of growth. Microinvasive Ефективність ДПТ раку ендометрія СОД 30 Гр на нашому матеріалі залежала ще і від глибини інвазії пухлини в міометрій, що відображено в таблиці 6.
tumors also have the lowest mitotic index, the most pronounced degree of cellular stromal reactions and the highest index of apoptosis. In tumors with invasion of the myometrium reaching more than 1.0 cm, the degree of radiation damage is the lowest, as well as the level of cellular stromal reactions. A moderate index of apoptosis in invasive tumors may be associated with the fact of being exposed to radiation as well as the factors that act on body level.
Thus, preoperative RRT of endometrial cancer by TFD 30 Gy leads to moderate regression of tumors with loss of 56 % of its volume, moderate dystrophy of the remaining TC, reduction of TC mitotic activity by 1.72 times compared to intact tumors. One patient (9.1 %) shows full regression and two patients (18.2 %) show rather significant regression of tumors after RRT.
Indices of radiation pathomorphosis of EC patients of Group II are presented in Table 7.

The outcomes presented in
Thus, preoperative RRT of endometrial cancer by TFD 30 Gy with radiomodification provides a rather positive effect and regression of more than 50.0 % of the tumor volume, reducing its biological potential and maintaining cellular immune responses. Besides, in two cases there was almost full regression of the tumor. Only 12.5 % remained and traces of 0.5 % of the tumor mass (Fig. 2).
The dependence of EC combined treatment outcomes on the histological structure and differentiation degree is known. It is believed that a malignant tumor (MT) with high proliferative potencies and low-grade differentiation degree is characterized by significant radiosensitivity, but in practice, this is not always the case, due to many properties of the tumor itself.
Chemoradiationpathomorphosis of EC patients of Group II depending on the histological structure of the tumor is presented in Table 8.
The obtained results indicate the highest regression of undifferentiated EC and full suppression of its proliferative activity, which corresponds to the literature data [29]. The lowest mitotic index was revealed in highly differentiated adenocarcinomas (1.50 ± 0.14‰). Proliferative activity is rather suppressed in low-and moderately differentiated endometrial adenocarcinomas, in comparison with intact tumors (7.86 ± 1.26 ‰).
TC dystrophy degree varied greatly within one histological tumor form as well as in tumors of different histological structure. However, the most pronounced degree of dystrophic changes in TC is observed in lowgrade differentiated adenocarcinoma.
The highest apoptosis index was observed in undifferentiated tumors (3.50 ± 0.99 %). In other words, inhibiting proliferative activity of tumors is important in the mechanism of regression.
Indices of radiation pathomorphosis of EC patients of Group II depending on tumor growth form are presented in Table 9.
Мінімальний ступінь ушкодження пухлини на тканинному рівні виявлений при їх екзофітно-ендофітній формі зростання. При цій формі зростання виявляється самий незначний ступінь ушкодження пухлини According to the data, the degree of radiation damage at the tissue level is greatest in exophytic tumors and in tumors localized within the mucosa. The dystrophy degree is also the highest in EC growth form. The lowest mitotic index is observed in exophytic tumors. The depth of invasion into the myometrium did not exceed 0.2 cm, i.e. it was minimal, ignoring the localization of the tumor within the mucosa.
in the TC and almost no regression at the tissue level. At the same time, the depth of invasion of such tumors into the myometrium is the largest, which indicates its high biological potential. Tumor regression at the tissue level and TC dystrophy are quite pronounced in tumors that have a low degree of invasion into the myometrium (Table 10).
Evidently, a moderate degree of radiation damage to the tumor at the tissue level is observed when the depth of tumor invasion is 0.5 cm and less. When this criterion is more than 0.5 cm, the degree of radiation damage at the tissue level is reduced by half. In our study, the most common cases were the ones characterized by a shallow invasion of the tumor into the myometrium, and the invasion depth reaching 1.2 cm was the deepest.
The degree of dystrophic changes in TC varied; it was the most pronounced in superficial tumors. In the tumors with deep invasion, a moderate degree of TC dystrophy is common, because in this part they are subjected to necrosis due to imperfect angiogenesis, and it is typical for the poorly differentiated tumors to grow so deep.
The mitotic index after RRT with radiomodification TFD 30 Gy is the lowest in superficial tumors along with the largest number of pathological mitoses and a high degree of cellular and immune stromal reactions. The apoptosis index is always the highest in tumors with a lowgraded differentiation and a high malignancy degree. It stands to reason that, the deepest invasion of the myometrium is typical for fast-growing tumors with a high malignancy level. At the same time, chemoradiation has an antiblastoma effect on such tumors reducing proliferative properties. The mitotic index in tumors with deep invasion of the myometrium was 1.5 times lower than in intact ones (7.87 ± 1.26 ‰).
Thus, preoperative RRT with radiomodification of TFD 30 Gy leads to a moderate degree of radiation damage to the tumor at the tissue level and decreased TC proliferative activity.
Таким чином, на підставі проведеного дослідження можна зробити виладені нижче висновки. effective in undifferentiated cancer cases. In tumor regression, apoptosis occurs only in undifferentiated tumors, but its role, according the level of the apoptosis index, is insignificant. Exophytic tumors and those limited by the mucous membrane are mostly sensitive to radiation exposure. Tumors with a large depth of invasion into the myometrium and exophytic-endophytic form of growth are minimally sensitive to radiation therapy. However, regardless of the depth of invasion in tumors, proliferative potencies are suppressed as well as mitotic is decreased, despite the low degree of radiation damage to the tumor at the tissue level.
Additionally, morphological examination of EC patients of Group I and Group II showed that the largest number of tumors was represented by moderately differentiated endometrial adenocarcinoma. In 2 cases, chemoradiation caused such a significant regression of the tumor that its residual volume in one case was 12.5 %, and in another one it was 0.5 %, which to some extent was still an evidence of a minor upside of radiomodification. When comparing tumor radiosensitivity depending on the histological structure, we did not find any significant differences.
A comparative assessment of radiation pathomorphosis in patients of the studied groups (Table 11) has revealed that when administering preoperative RRT course of 30 Gywith radiomodification, there is a probable decrease in the mitotic index (p < 0.01) (4.58 ± 1.10 and 3.14 ± 0.73 ‰, respectively), in comparison with Group 3. Such markers as tumor regression degree, number of pathological mitoses, nature of stromal reactions tend to be increased during RRT preoperative course of TFD 30 Gy with radiomodification.
Thuswise, the study has made it possible to conclude the following.