Оценка васкуляризации и микроокружения опухолевой ткани при фолликулярной лимфоме

Нестерова Е.С., Кравченко С.К., Гемджян Э.Г., Османов Е.А., Ковригина А.М. Оценка васкуляризации и микроокружения опухолевой ткани при фолликулярной лимфоме. Терапевтический архив (архив до 2018 г.). 2013;85(7):57-64.

Читать PDF

Аннотация

Цель исследования. Оценить васкуляризацию и микроокружение опухолевой ткани при фолликулярной лимфоме (ФЛ) на основе иммуногистохимического исследования (ИГХИ) на срезах с парафиновых блоков биоптатов лимфатических узлов в сравнительном исследовании 2 групп пациентов, отличающихся исходами заболевания.
Материалы и методы. В исследование включили 59 больных (39 (67%) женщин и 20 (33%) мужчин) в возрасте от 27 до 83 лет (медиана 53 года), проходивших лечение с апреля 2001 г. по май 2011 г. в Гематологическом научном центре МЗ РФ (n=49) и в Российском онкологическом научном центре им. Н.Н. Блохина РАМН (n=10). В соответствии с особенностями клинического течения заболевания выделены 2 группы пациентов: 1-я (n=31) — с хорошими результатами лечения ФЛ и 2-я (n=28) — с неблагоприятными исходами. ИГХИ выполнено на срезах с парафиновых блоков биоптатов опухолевых лимфатических узлов до лечения с использованием антител к CD34, D2-40, CD68 и granzyme B. Морфометрический анализ проведен с использованием микроскопии и цифровой камеры Leica (об. ×400). Фотографии гистологических препаратов обработаны с помощью компьютерной программы «ВидеоТесТ-Морфология 5.2»: оценена удельная площадь сосудов (в процентах) по отношению к опухолевой ткани при визуальном контроле исследователя. Количественная характеристика цитотоксических лимфоцитов (ЦТЛ) и макрофагов проводилась на 1 мм2 опухолевой ткани (12 полей зрения при увеличении объектива ×400). Выбор иммуногистохимических препаратов для исследования проводили случайным образом (с помощью таблицы случайных чисел).
Результаты. Удельная площадь кровеносных сосудов во 2-й группе оказалась статистически значимо больше, чем в 1-й: 0,04% (при 95% доверительном интервале — ДИ от 0,03 до 0,05%) против 0,02% (при 95% ДИ от 0,01 до 0,03%; р=0,05). Удельная площадь лимфатических сосудов во 2-й группе значимо больше, чем в 1-й: 0,06% (при 95% ДИ от 0,04 до 0,07%) против 0,03% (при 95% ДИ от 0,01 до 4%; p=0,03). При нодулярно-диффузном характере роста во 2-й группе количество CD68-позитивных макрофагов оказалось значимо больше, чем в 1-й: 800 (при 95% ДИ от 380 до 1222) против 79 (при 95% ДИ от 10 до 566; р=0,01). Количество ЦТЛ в 1-й группе статистически значимо (р=0,05) больше, чем во 2-й, как в нодулярном (при нодулярном росте опухоли: 14 (5—27) против 5 (1—11)), так и в интернодулярном пространстве (при нод улярном характере роста опухоли: 158 (118—410) против 35 (5—287) и при нодулярно-диффузном характере роста: 126 (102—360) против 35 (3—120)).
Заключение. Повышенная васкуляризация опухоли (оцениваемая удельной плотностью сосудистой сети в опухолевой ткани) и выраженная макрофагальная реакция ассоциированы с неблагоприятными исходами фолликулярной лимфомы; выраженный цитотоксический компонент в опухолевой ткани ассоциирован с благоприятными исходами заболевания.

Ключевые слова: фолликулярная лимфома, кровеносные сосуды, лимфатические сосуды, макрофаги, цитотоксические лимфоциты, поисковый анализ данных, многофакторный статистический анализ.

________________________________________________

Aim. To characterize the degree of follicular lymphoma (FL) vascularization and microenvironment by immunohistochemical studies (IHCS) of lymph node biopsy paraffin-embedded sections in 2 different disease pattern groups.
Subjects and methods. The investigation included 59 patients: 39 (67%) women and 20 (33%) men whose age was 27 to 83 years (median age 53 years) treated at the Hematology Research Center, Ministry of Health of the Russian Federation (n=49), and the N.N. Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences (n=10), in April 2001 to May 2011. In accordance with the clinical features of the disease, the authors identified 2 patient groups: 1) 31 patients with the good results of FL treatment and 2) 28 patients with its poor results. IHCS was performed on lymph node tumor biopsy paraffin-embedded sections prior to treatment using antibodies to CD34, D2-40, CD68, and granzyme B. Morphometric analysis was made applying microscopy and a Leica ×400 digital camera. The images of histological specimens were processed by the computer program VideoTesT-Morphology 5.2: the specific vessel area (%) in relation with tumor tissue was estimated under visual guidance of an investigator. Cytotoxic lymphocytes (CTL) and macrophages were quantitatively characterized using 1 mm2 of tumor tissue (12 fields of vision with the objective lens magnifying ×400). Immunohistochemical specimens to be examined were chosen randomly, by using the random number table.
Results. In Group 2, the specific area of blood vessels was statistically significantly higher than in Group 1: 0.04% (95% confidence interval (CI), 0.03 to 0.05%) versus 0.02% (95% CI, 0.01 to 0.03%; p=0.05). In Group 2, that of lymphatic vessels was significantly higher than in Group 1: 0.06% (95% CI, 0.04 to 0.07%) versus 0.03% (95% CI, 0.01 to 4%; p=0.03). With a nodular diffuse growth, Group 2 showed a significantly more CD68-positive macrophages than did Group 1: 800 (95% CI, 380 to 1222) versus 79 (95% CI, 10 to 566; р=0.01). In Group 1, the count of CTL was statistically significantly (p=0.05) higher than in Group 2 in both the nodule (with a nodular growth pattern: 14 (5-27) versus 5 (1—11)) and the internodular space (with a nodular growth pattern: 158 (118—410) versus 35 (5—287) and with a nodular diffuse growth pattern: 126 (102—360) versus 35 (3—120)).
Conclusion. Increased tumor vascularization (estimated by the specific density of tumor vasculature) and a pronou nced macrophageal reaction are associated with the poor outcomes of FL; the marked cytotoxic component in tumor tissue is linked to the favorable outcomes of the disease.

Key words: follicular lymphoma, blood vessels, lymphatic vessels, macrophages, cytotoxic lymphocytes, exploratory data analy-sis, multivariate statistical analysis.

Список литературы

Morton L.M., Wang S.S., Devesa S.S. et al. Lymphoma incidence patterns by WHO subtype in the United States, 1992-2001. Blood 2006; 107 (1): 265-276.
A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma. The Non-Hodgkin's Lymphoma Classification Project. Blood. 1997; 89 (11): 3909-3918.
Rohatiner A.Z., Nadler L., Davies A.J. et al. Myeloablative therapy with autologous bone marrow transplantation for follicular lymphoma at the time of second or subsequent remission: long-term follow-up. J Clin Oncol 2007; 25 (18): 2554-2559.
van Oers M.H., Van Glabbeke M., Giurgea L. et al. Rituximab maintenance treatment of relapsed/resistant follicular non-Hodgkin's lymphoma: long-term outcome of the EORTC 20981 phase III randomized intergroup study. J Clin Oncol 2010; 28 (17): 2853-2858.
Schouten H.C., Qian W., Kvaloy S. et al. High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin's lymphoma: results from the randomized European CUP trial. J Clin Oncol 2003; 21 (21): 3918-3927.
Hazar B., Paydas S., Zorludemir S. et al. Prognostic significance of microvessel density and vascular endothelial growth factor (VEGF) expression in non-Hodgkin's lymphoma. Leuk Lymphoma 2003; 44: 2089-2093.
Tzankov A., Heiss S., Ebner S. Angiogenesis in nodal B cell lymphomas: a high throughput study. J Clin Pathol 2007; 60: 476-482.
Ferrara N., Winer J., Burton T. et al. Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells. J Clin Invest 1993; 91: 160-170.
Zhang H.T., Craft P., Scott P.A. et al. Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells. J Nat Cancer Inst 1995; 87: 213-219.
Murrell J.C., Lidstrom M.E., Holmes A.J., Costello A. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett 1995; 132 (3): 203-208.
Yamaguchi H., Takagi J., Miyamae T. et al. Milk fat globule EGF factor 8 in the serum of human patients of systemic lupus erythematosus. J Leukoc Biol 2008; 83 (5): 1300-1307.
Barleon B., Sozzani S., Zhou D. et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1 Blood 1996; 87: 3336-3343.
Hiratsuka S., Minowa O., Kuno J. et al. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci USA 1998; 95: 9349-9354.
Yang Z.F., Poon R.T., Luo Y. et al. Up-regulation of vascular endothelial growth factor (VEGF) in small-for-size liver grafts enhances macrophage activities through VEGF receptor 2-dependent pathway. J Immunol 2004; 173: 2507-2515.
Skobe M., Hamberg L.M., Hawighorst T. et al. Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am J Pathol 2001; 159: 893-903.
Pepper M.S. Lymphangiogenesis and Tumor Metastasis: Myth or Reality? Clin Cancer Res 2001; 7 (3): 462-468.
Schneider M., Othman-Hassan K., Christ B., Wilting J. Lymphangioblasts in the avian wing bud. Dev Dyn 1999; 216: 311-319.
Takahashi T., Kalka C., Masuda D. et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999; 5: 434-438.
Gill M., Dias K., Hattori M.L. et al. Vascular trauma induces rapid but transient mobilization of VEGFR2(+) AC133(-) endothelial precursor cells. Circ Res 2001; 88: 167-174.
Dengjel J., Kratchmarova I., Blagoev B. Receptor tyrosine kinase signaling: a view from quantitative proteomics. Mol Biosyst 2009; 5 (10): 1112-1121.
Folkman J., D'Amore P.A. Blood vessel formation: what is its molecular basis? Cell 1996; 87: 1153-1155.
Holmes K., Owain R.L., Angharad M.T. Cross M.J. Vascular endothelial growth factor receptor-2: Structure, function, intracellular signalling and therapeutic inhibition. Cellular Signalling 2007; 19 (10): 2003-2012.
Wey J.S., Fan F., Gray M.J. et al. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer 2005; 104: 427-438.
Koster A., Raemaekers J.M. Angiogenesis in malignant lymphoma. Curr Opin Oncol 2005; 17: 611-616.
Foss H.D., Araujo I., Demel G. et al. Expression of vascular endothelial growth factor in lymphomas and Castleman's disease. J Pathol 1997; 183: 44-50.
Ribatti D., Vacca A., Bertossi M. et al. Angiogenesis induced by B-cell non-Hodgkin's lymphomas. Lack of correlation with tumor malignancy and immunologic phenotype. Anticancer Res 1990; 10: 401-406.
Stewart M., Talks K., Leek R. et al. Expression of angiogenic factors and hypoxia inducible factors HIF 1, HIF 2 and CA IX in non-Hodgkin's lymphoma. Histopathology 2002; 40: 253-260.
Jørgensen J.M., Sørensen F.B., Bendix K. Expression level, tissue distribution pattern, and prognostic impact of vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4 in different subtypes of non-Hodgkin lymphomas. Leuk Lymphoma 2009; 50 (10): 1647-1660.
Ho C.L., Sheu L.F., Li C.Y. Immunohistochemical expression of angiogenic cytokines and their receptors in reactive benign lymph nodes and non-Hodgkin lymphoma. Ann Diagn Pathol 2003; 7 (1): 1-8.
Gately S., Li W.W. Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy. Semin Oncol 2004; 31 (Suppl 7): 2-11.
Hoper M.M., Voelkel N.F., Bates T.O. et al. Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP. Am J Respir Cell Mol Biol 1997; 17: 748-756.
Bellamy W.T., Richter L., Frutiger Y. et al. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res 1999; 59: 728-733.
Bono P., Teerenhovi L., Joensuu H. Elevated serum endostatin is associated with poor outcome in patients with non-Hodgkin lymphoma. Cancer 2003; 97: 2767-2775.
Pedersen L.M., Klausen T.W., Davidsen U.H. et al. Early changes in serum IL-6 and VEGF levels predict clinical outcome following first-line therapy in aggressive non-Hodgkin's lymphoma. Ann Hematol 2005; 84: 510-516.
Potti A., Ganti A.K., Kargas S. et al. Immunohistochemical detection of C-kit (CD117) and vascular endothelial growth factor (VEGF) overexpression in mantle cell lymphoma. Anticancer Res 2002; 22: 2899-2901.
Salven P., Orpana A., Teerenhovi L. et al. Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma: a singleinstitution study of 200 patients. Blood 2000; 96: 3712-3718.
Vacca A., Ribatti D., Ruco L. et al. Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas. Br J Cancer 1999; 79: 965-970.
Zhao W.L., Mourah S., Mounier N. et al. Vascular endothelial growth factor-A is expressed both on lymphoma cells and endothelial cells in angioimmunoblastic T-cell lymphoma and related to lymphoma progression. Lab Invest 2004; 84: 1512-1519.
Koster A., van Krieken J.H., Mackenzie M.A. et al. Increased vascularization predicts favorable outcome in follicular lymphoma. Clin Cancer Res 2005; 11: 154-161.
Aggarwal D., Srivastava G., Gupta R. et al. Angiogenesis in Non-Hodgkin's Lymphoma: An Intercategory Comparison of Microvessel Density. ISRN Hematol 2012; 2012: 943089.
Farinha P., Kyle A.H., Minchinton A.I. et al. Vascularization predicts overall survival and risk of transformation in follicular lymphoma. Haematologica 2010; 95 (12): 2157-2160.
Shayan R., Achen M.G., Stacker S.A. Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 2006; 27 (9): 1729-1738.
Banerji S., Ni J., Wang S.X. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 1999; 144: 789-801.
Wigle J.T., Oliver G. Prox1 function is required for the development of the murine lymphatic system. Cell 1999; 98: 769-778.
Wahlin B.E., Aggarwal M., Montes-Moreno S. A Unifying Microenvironment Model in Follicular Lymphoma: Outcome Is Predicted by Programmed Death-1-Positive, Regulatory, Cytotoxic, and Helper T Cells and Macrophages. Clin Cancer Res 2010; 16 (2): 637-650.
Mosser D.M. The many faces of macrophage activation. J Leukoc Biol 2003; 73 (2): 209-212.
Stix G. A malignant flame. Understanding chronic inflammation, which contributes to heart disease, Alzheimer's and a variety of other ailments, may be a key to unlocking the mysteries of cancer. Sci Am 2007; 297 (1): 60-67.
Jong D., Koster A., Hagenbeek A. et al. Impact of the tumor microenvironment on prognosis in follicular lymphoma is dependent on specific treatment protocols. Haematologica 2009; 94: 70-77.
Richendollar B.G., Pohlman B., Elson P., Hsi E.D. Follicular programmed death 1-positive lymphocytes in the tumor microenvironment are an independent prognostic factor in follicular lymphoma. Hum Pathol 2011; 42 (4): 552-557.
Clear A.J., Lee A.M., Calaminici M. et al. Increased angiogenic sprouting in poor prognosis FL is associated with elevated numbers of CD163-macrophages within the immediate sprouting microenvironment. Blood 2010; 115 (24): 5053-5056.
Andjelic B., Mihaljevic B., Todorovic M. et al. The number of lymphoma-associated macrophages in tumor tissue is an independent prognostic factor in patients with follicular lymphoma. Appl Immunohistochem Mol Morphol 2012; 20 (1): 41-46.
Álvaro T., Lejeune M., Camacho F.I. et al. The presence of STAT1-positive tumor-associated macrophages and their relation to outcome in patients with follicular lymphoma. Haematologica 2006; 91: 1605-1612.
Canioni D., Salles G., Mounier N. et al. High Numbers of Tumor-Associated Macrophages Have an Adverse Prognostic Value That Can Be Circumvented by Rituximab in Patients With Follicular Lymphoma Enrolled Onto the GELA-GOELAMS FL-2000 Trial. J Clin Oncol 2008 26 (3): 440-446.
Taskinen M., Karjalainen-Lindsberg M.-L., Nyman H. et al. A HighTumor-Associated Macrophage Content Predicts Favorable Outcome in Follicular Lymphoma Patients Treated with Rituximab and Cyclophosphamide-Doxorubicin-Vincristine-Prednisone. Clin Cancer Res 2007; 13: 5784-5789.
Leger-Ravet M.B., Devergne O., Peuchmaur M. In situ detection of activated cytotoxic cells in follicular lymphomas. Am J Pathol 1994; 144 (3): 492-499.
Alvaro T., Lejeune M., Salvado M.T. et al. J Clin Oncol 2007; 25 (10): 1289-1891.
Lee A.M., Clear A.J., Calaminici M. et al. Number of CD4 Cells and Location of Forkhead Box Protein P3-Positive Cells in Diagnostic Follicular Lymphoma Tissue Microarrays Correlates With Outcome. J Clin Oncol 2006; 24 (31): 5052-5059.
Laurent C., Müller S., Do C. et al. Distribution, function, and prognostic value of cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study. Blood 2011; 118 (20): 5371-5379.
Нестерова Е.С., Кравченко С.К., Гемджян Э.Г. и др. Лечение фолликулярной лимфомы: 10-летний опыт. Гематол и трансфузиол 2012; 5: 3-8.
Покровская О.С., Менделеева Л.П., Капланская И.Б. и др. Ангиогенез в костном мозге больных множественной миеломой на различных этапах высокодозной химиотерапии. Клин онкогематол 2010; 4: 347-353.
Любимова Л.С., Савченко В.Г., Менделеева Л.П. и др. Трансплантация аллогенного костного мозга при хроническом миелолейкозе. Тер арх 2004; 7: 18-24.
Пивник А.В., Самойлова Р.С., Новиков В.А. и др. Дифференциально-диагностическое и прогностическое значение показателей сывороточных цитокинов при лимфогранулематозе и лимфосаркомах. Тер арх 2001; 4: 45-51.
Магомедова А.У., Кравченко С.К., Кременецкая А.М. и др. Модифицированная программа NHL-BFM-90 для лечения больных диффузной В-крупноклеточной лимфосаркомой. Тер арх 2006; 10: 44-47.
Магомедова А.У., Кравченко С.К., Кременецкая А.М. и др. Эффективность курса CHOP-21 в терапии диффузной В-крупноклеточной лимфосаркомы. Тер арх 2005; 7: 58-61.
Swerdlow S.H., Campo E., Harris N.L. et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008.

Авторы

Е.С. НЕСТЕРОВА 1, С.К. КРАВЧЕНКО 1, Э.Г. ГЕМДЖЯН 1 , Е.А. ОСМАНОВ 2, А.М. КОВРИГИНА 1

1 Гематологический научный центр Минздрава России;
2 Российский онкологический научный центр им. акад. Н.Н. Блохина РАМН, Москва

________________________________________________

E.S. NESTEROVA 1, S.K. KRAVCHENKO 1, E.G. GEMDJIAN 1, E.A. OSMANOV 2, A.M. KOVRIGINA 1

1 Hematology Research Center, Ministry of Health of Russia;
2 Acad. N.N. Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Moscow

Назад к списку

Цель портала OmniDoctor – предоставление профессиональной информации врачам, провизорам и фармацевтам.