ЭКСПРЕССИЯ МАРКЕРОВ CD44 И CD24 В БИОПСИЙНОМ МАТЕРИАЛЕ БОЛЬНЫХ ТРОЙНЫМ НЕГАТИВНЫМ РАКОМ МОЛОЧНОЙ ЖЕЛЕЗЫ ДО ЛЕЧЕНИЯ

The role of the expression of CD44 and CD24 in breast cancer (BC) has been explored in many laboratories around the world to identify predictive markers of tumor aggressiveness and patient’s response to anticancer therapy. These proteins participate in the process of tumor growth, metastasis and formation of cancer stem cells (CSCs). The study of CD44 and CD24 expression in triple negative (TN) BC, which is the most aggressive breast cancer subtype, is of particular interest. The aim of this study was to determine the relationship between the expression of CD44 and CD24 markers in biopsy samples of TNBC patients before treatment and clinical/ morphological characteristics of the tumors. Material and Methods. The study group included 67 patients with stage I–IV TNBC. Flow cytometry was used to determine the proportion of cells with CSC immunophenotype (CD44+/CD24-/low) in biopsy samples from the primary tumor of 65 patients and lymph nodes of 6 patients. In addition, the proportion of cells with all possible combinations of expression of these surface proteins was estimated. Results. Cells with CSC immunophenotype were detected in all patients with a wide individual variability of CSC proportion from 0.4 % to 77.0 % (median – 10.9 %). There were no differences in the proportion of CSCs in the primary tumor and lymph nodes. No statistically significant correlation between the proportion of CSCs in the primary tumor and the clinical/morphological parameters, including tumor size and differentiation grade, evidence of regional or distant metastases, tumor, size of the fraction of proliferating cells estimated by Ki67 expression, was found in either single or multivariate analysis. There was also no association of the above parameters (except Ki67) with immunophenotypes. A high proportion of Ki67-positive cells in the primary tumor was associated with the CD44-CD24-phenotype. Conclusion. The expression of CD44 and CD24 in biopsy samples of TNBC before treatment did not correlate with the clinical and morphological characteristics of the tumors, excepting Ki67 expression.Индивидуальные особенности экспрессии CD44 и CD24 при раке молочной железы (РМЖ) изучаются во многих лабораториях мира в рамках поиска прогностических маркеров агрессивности опухолевого процесса и эффективности противоопухолевой терапии, что во многом обусловлено участием этих белков в процессах опухолевого роста, метастазирования и формирования популяции опухолевых стволовых клеток (ОСК), которые составляют наиболее резистентную часть злокачественных новообразований к радиационным и многим химиотерапевтическим воздействиям. Особый интерес представляет исследование экспрессии CD44 и CD24 в случае тройного негативного (ТН) РМЖ – наиболее агрессивного среди различных молекулярных подтипов злокачественных новообразований данной локализации. Цель исследования – выяснение возможной взаимосвязи экспрессии маркеров CD44 и CD24 в биопсийном материале больных ТН РМЖ до лечения с клинико-морфологическими характеристиками опухоли. Материал и методы. В исследование включено 67 больных ТН РМЖ I–IV стадии. С помощью проточной цитометрии в биоптатах из первичного опухолевого очага 65 больных и лимфоузлов 6 больных определяли долю клеток с иммунофенотипом ОСК, которые характеризуются наличием на клеточной мембране CD44 при низкой экспрессии CD24 или отсутствии таковой (CD44+/ CD24-/low). Кроме того, оценивали долю клеток со всеми возможными комбинациями экспрессии этих поверхностных белков. Результаты. Клетки с иммунофенотипом ОСК выявлены в первичном очаге всех больных при широкой индивидуальной вариабельности их доли – от 0,4 до 77,0 % (медиана – 10,9 %). Не обнаружено различий доли ОСК в первичном очаге и в лимфоузлах. Ни при одно-, ни при многофакторном анализе не установлено значимой корреляции доли ОСК ни с одним из клинико-морфологических показателей, включая размер и степень дифференцировки опухоли, наличие регионарных и отдаленных метастазов, размер фракции пролиферирующих клеток, оцениваемый по экспрессии Ki67. Также не установлено ассоциации указанных показателей (кроме Ki67) ни с одним из преобладающих в исследованном материале иммунофенотипов. В первичном опухолевом очаге высокая доля Ki67-позитивных клеток была ассоциирована с фенотипом CD44-CD24-. Заключение. Экспрессия маркеров CD44 и CD24 в клетках биопсийного материала больных ТН РМЖ до лечения не коррелирует с клинико-морфологическими характеристиками опухоли, кроме экспрессии Ki67

Системная фотодинамическая терапия с фотосенсибилизатором фотолон в лечении онкологических больных с регионарными и отдаленными метастазами

 The results of photodynamic therapy (PDT) in 76 patients with tumors of different sites and with regional or distant metastases are represented. Sixty three patients were under combined or multimodal therapy, 13 patients had systemic PDT as monotherapy. The technique of PDT was as follows: the solution of photosensitizer photolon was administered intravenously at dose of 0.8–1.4 mg/ kg body weight. Laser blood irradiation was performed simultaneously (wavelength of 662 nm, output power of 20 mW, irradiation time of 50 min). Stabilization of the disease defined as the absence of new tumor foci was observed in 55% of treated patients: among them in 47% of patients with disseminated melanoma during 6–10 months after treatment, in 65% patients with breast cancer – for 3–6 years after treatment, and also in 100% of patients with cancer of other sites (colorectal, pancreatic, cervical, ovarian, lung and stomach cancer, retroperitoneal neuroblastoma) – for 10–12 months after treatment. According to ultrasound data the shrinkage of most of metastases up to its complete disappearance was observed. The authors consider that effects of systemic PDT are due to decrease of circulating tumor cells in blood and also due to beneficial impact of this modal of treatment on immune status of cancer patient. Intravenous PDT was shown to improve treatment results and quality of life in patients with metastases of malignant tumors. The approved technique is of considerable interest and requires further investigation of its efficiency including its combination with methods of combined and multimodal treatment. Приведены результаты применения фотодинамической терапии (ФДТ) у 76 пациентов с опухолями различной локализации и наличием регионарных или отдаленных метастазов. Шестьдесят три пациента находились в процессе комбинированного или комплексного лечения, 13 пациентам системная ФДТ проводилась как монотерапия. Методика проведения ФДТ заключалась в следующем: внутривенно вводили раствор фотосенсибилизатора фотолон в дозе 0,8–1,4 мг/кг массы тела. Одновременно проводили лазерное облучение крови больного (длина волны 662 нм, мощность лазера на выходе 20 мВт, время облучения 50 мин). Стабилизация процесса в виде отсутствия новых очагов опухоли наблюдалась у 55% пролеченных пациентов: в том числе у 47% больных с диссеминированной меланомой в течение 6–10 мес. после лечения, у 65% больных раком молочной железы – в течение 3–6 лет после лечения, а также у 100% больных с онкологической патологией другой локализации (рак прямой и ободочной кишки, поджелудочной железы, шейки матки, яичников, легкого и желудка, нейробластома забрюшинного пространства) – в течение 10–12 мес. после лечения. По данным УЗИ, отмечено уменьшение размеров большей части метастатических очагов вплоть до их полного исчезновения. Авторы считают, что реализация эффектов системной ФДТ происходит за счет уменьшения циркулирующих в крови опухолевых клеток, а также вследствие положительного влияния данного вида лечения на иммунный статус онкологических больных. Показано, что применение внутривенной ФДТ позволяет улучшить результаты лечения и качество жизни больных с метастазами злокачественных новообразований. Апробированная методика представляет значительный интерес, что требует дальнейшего изучения ее эффективности, в том числе, в сочетании с методами комбинированного и комплексного лечения.

Individual Response to Low Dose Radiation Exposure as Determined by TCR Assay

The aim of the work was to study the level of somatic gene mutagenesis in various human contingents exposed to ionizing radiation at doses up to 200 mSv. Flow cytometry was used to determine frequency of variant CD4+ T-lymphocytes with altered T-cell receptor (TCR) /CD3 expression as result of mutations at TCR locus. The TCR-mutant cell frequency was assessed in 250 unexposed control donors and 1198 exposed persons, including employees of Nuclear Power Engineering (mean dose (±SE) 100,0±6,6 MSv was accumulated within 27,4±1,1 years), cleanup workers of the Chernobyl accident (mean dose 100,0±9,1 MGy, analysis was performed 9-19 years after the irradiation) and persons living in radiation contaminated areas (137Cs density 37- 555 kBq/m2) during 15-19 years. Results of group analysis demonstrated an increase in frequency of the TCR-mutant cells in NPE employees and the Chernobyl cleanup workers compared to that in age-matched control groups (p<0.05, Mann-Whitney test). About 18 % of persons in both exposed groups had the TCRmutant cell frequencies exceeding the 95% confidence interval that had been determined in control groups. Distribution of the mutant cell frequencies in other exposed persons corresponded to that in control group. The proportion of persons with elevated mutant cell frequencies was inversely proportional to age at exposure and did not depend on dose. Elevation of the mutant cell frequency significantly correlated with levels of intracellular NO and apoptosis of lymphocytes after additional irradiation in vitro. The frequency of the TCR-mutant cells in residents of radiation contaminated areas was significantly higher than in age-matched control groups (p<0.05, Mann-Whitney test). Radiation effect depended on developmental stage at the moment of beginning of exposure. The most pronounced elevation of the TCR-mutant cell frequency was found in the individuals irradiated in utero. Only a proportion (12%) of persons exposed in postnatal period had the TCRmutant cell frequencies exceeding the 95% confidence interval in the control group. The proportion of the persons with increased TCR-mutant cell frequencies was higher among residents of areas contaminated with 137Cs density over 100 kBq/m2 in comparison to less contaminated areas: 14,5% vs 8,8% accordingly (p=0,02, Fishers test). In conclusion, relatively small proportion of persons was found to react to low dose radiation exposure by elevation of the TCR-mutant cell frequency in all groups excluding irradiation in utero. The work was supported by Russian Foundation of Basic Research (# 08-04-00790)

The Emergency Workers Medical Observation at the Long Time after the Chernobyl NPP Accident

The approximately 500 emergency workers (liquidators) have been observed in the Treatment and Diagnostic center (Obninsk, Russia). The majority of them were men worked in Chernobyl NPP area in 1986. The total dose of external irradiation was up to 250 mSv. The basic pathology was cardiovascular diseases: ischemic heart disease, hypertension, multifocal atherosclerosis. Associated diseases were osteochondrous, bronchitis, goitre, obesity, prostatitis. In some cases (N=6) the oncopathology has been estimated (thyroid cancer 2, prostate cancer 2, kidney cancer 1, stomach cancer 1). The frequency of lymphocytes bearing mutated T-cell receptors was assessed in 184 liquidators at the long time after the Chernobyl accident. The mutant cell frequency was elevated in 31 patients of this group as compared with control group. We have not found any difference in the clinical data and the somatic diseases manifestation among liquidators and other nonexposed patients. The treatment of liquidators undergoes according to the modern approaches. The emergency workers demonstrated the alterations in the psychological status: high level of personal anxiety, hesitation, indecision, tension, pressure, inner conflict, the reducing of social activity. The MMPI profile (multifactor method of personal identification) of liquidators with cerebrovascular diseases was characterized by the mixed response with the hypochondria prevalence. The indices of the hypochondria, depression, hysteria and psychoastenia scales in the liquidators group were significantly higher than in the control group patients with the same diseases but had no contact with ionizing radiation. Cerebrovascular diseases presence was not the main reason of the psychological status abnormalities in liquidators. The prolonged psychological impact of the Chernobyl accident caused somatic complains and disease-orientated behavior of liquidators and resulted in reducing their social activity. Thus the medical screening of emergency workers and the special psychological support of them should be continued. The special medical observation of liquidators with the elevated level of mutant cell frequency should be prolonged because of possible high cancer risk in this group

Genomic instability in mice long term after radiation exposure at low dozes

In this study we measured frequency of lymphocytes bearing mutation at T-cell receptor locus (TCR) and frequency of micronucleated erythrocytes in peripheral blood of mice. Experiments were conducted with male CBA strain of mice which irradiated at 0,2 Gy of γ-rays using Co60. Blood sampling was made 3 and 6 months after the irradiation. As it was shown with Mann-Whitney test the frequencies of TCR-mutant cells in exposed and control unexposed groups were not significantly different (p>0,05). According to results of 3 independent experiments medians of TCR mutant cell frequency were 11,7 x 10 -4 . in the controls and 12,5 x 10-4 in exposed group 3 months after the irradiation (p>0,05). Medians increased slightly in both groups 6 months after radiation exposure: 13,8 x 10-4 and 15,5 x 10 -4 in control and exposed groups accordingly (p> 0,05). 95% confidence intervals for TCR mutant cell frequency were calculated in control groups. About 15 % of exposed mice had frequencies of the mutant cells exceeding upper boundary of this interval 3 month after the irradiation. The proportion of exposed mice with high TCR mutant cell frequencies increased up to 21% 6 months after the irradiation (p<0,05 in comparison to control group, Fisher’s test). Mean frequencies of micronuclei in normochromic erythrocytes of peripheral blood of control and exposed mice were not significantly different 3 and 6 months after irradiation. In addition we estimated frequency of micronucleated reticulocytes in number of mice with the high frequency of TCR-mutant cells 6 months after the irradiation. For the control we took 6 unexposed mice that had casually been chosen on the same term. Mean frequencies of micronucleated reticulocytes were not different: (3,1±1,1) x 10-3 and (3,2±1,0) x 10-3 in control and exposed groups accordingly. Thus, genomic instability over 3 and 6 months after the irradiation was observed in proportion of mice on gene level and was not found on chromosomal level. It is arguable that the mechanisms underlying formation of genomic instability after low-LET radiation exposure at low doses in vivo involve, as a rule, only little changes in DNA structure (single-strand break, base substitution, etc.), that lead to formation of mainly gene mutations. It is possible also, that elimination of cells with micronuclei occurs more intensively, than cells with TCR mutations

Radiation Response of Cervical Cancer Stem Cells Is Associated with Pretreatment Proportion of These Cells and Physical Status of HPV DNA

Radio- and chemoresistance of cancer stem cells (CSCs) is considered as one of the possible causes of adverse results of chemoradiotherapy for various malignancies, including cervical cancer. However, little is known about quantitative changes in the CSC subpopulation in the course of treatment and mechanisms for individual response of CSCs to therapy. The purpose of the study was to evaluate the association of radiation response of cervical CSCs with clinical and morphological parameters of disease and features of human papillomavirus (HPV) infection. The proportion of CD44+CD24low CSCs was determined by flow cytometry in cervical scrapings from 55 patients with squamous cell carcinoma of uterine cervix before treatment and after fractionated irradiation at a total dose of 10 Gy. Real-time PCR assay was used to evaluate molecular parameters of HPV DNA. Post-radiation increase in the CSC proportion was found in 47.3% of patients. Clinical and morphological parameters (stage, status of lymph node involvement, and histological type) were not significantly correlated with radiation changes in the CSC proportion. Single- and multifactor analyses revealed two independent indicators affecting the radiation response of CSCs: initial proportion of CSCs and physical status of HPV DNA (R = 0.86, p = 0.001 for the multiple regression model in the whole)

Structure Formation and Tribological Properties of Mo-Si-B-Hf Electrospark Coatings Based on Mo2Ni3Si Laves Phase

Coatings were produced on the EP741NP nickel alloy substrates by electrospark deposition (ESD) in argon using an MoSi2-MoB-HfB2 electrode. In situ high-resolution transmission electron microscopy and X-ray diffraction analysis studies have identified the temperature above which the strengthening Mo2Ni3Si Laves phase is formed in the coatings. At 25 &deg;C, the coatings with a predominant content of the Laves phase are characterized by enhanced wear resistance, as well as a lower coefficient of friction compared to the non-annealed coatings containing binary silicides. At 700 &deg;C, the EP741NP substrate was characterized by the lowest friction coefficient (Ktr = 0.35), and its wear was approximately at the same level as the wear of both coatings