Діагностика анапластичних пухлин та пухлин без встановленої первинної локалізації: імуноморфологічні аспекти

Abstract

В дисертаційній роботі для покращення біопсійної діагностики недиференційованих (анапластичних) пухлин та метастазів пухлин з невідомого первинного джерела досліджено імуногістохімічні фенотипи та морфометричні характеристики «площа», «периметр» та «коефіцієнт круглості» шляхом обробки цифрових мікрофотографій зрізів програмою ІmageJ в пухлинах епітеліального, мезенхімального, меланоцитарного та гемолімфоцитарного походження. Визначені категорії верифікації анонімних метастатичних уражень для розуміння ефективності імуногістохімічного метода в діагностиці пухлин з невідомого джерела. Сформовані імуногістохімічні та морфометричні критерії для сприятливих імунофенотипів синдрому «Рак невідомої первинної локалізації». Визначено частку імунофенотипів синдрому «Рак невідомої первинної локалізації» з наявністю ампліфікації гену ERBB2 й делеції локусу 9p21 гену CDKN2A методом FISH та порівняно із відповідною зміною експресії протеїнів HER-2/neu та р16 ІNK4A. В диссертационной работе для улучшения биопсийной диагностики недифференцированных (анапластических) опухолей и метастазов опухолей из неизвестного первичного источника исследованы иммуногистохимические фенотипы и морфометрические характеристики «площадь», «периметр» и «коэффициент круглости» путем обработки цифровых микрофотографий срезов программой ІmageJ в опухолях эпителиального, мезенхимального, меланоцитарного и гемолимфоцитарного происхождения. Определены категории верификации анонимных метастатических поражений для понимания эффективности иммуногистохимического метода в диагностике опухолей неизвестного происхождения. Сформированы иммуногистохимические и морфометрические критерии для благоприятных иммунофенотипов синдрома «Рак неизвестной первичной локализации». Определена часть иммунофенотипов синдрома «Рак неизвестной первичной локализации» с наличием амплификации гена ERBB2 и делеции локуса 9p21 гена CDKN2A методом FISH в сравнении с соответствующим изменением экспрессии протеинов HER-2 / neu и р16 ІNK4A. In the dissertation work to improve the biopsy diagnosis of undifferentiated (anaplastic) tumors and tumor metastases from an unknown primary site, immunohistochemical (IHC) phenotypes and morphometric (MPM) characteristics of "area", "perimeter" and "round" were studied in tumors of epithelial, mesenchymal, melanocyte and hemolymphocyte origin; defined categories of verification of anonymous metastatic lesions to understand the effectiveness of the IHC method in the diagnosis of tumors from an unknown site; formed IHC and MPM criteria for favorable immunophenotypes of the syndrome "Cancer of unknown primary site" (CUP); a part of CUP with the presence of ERBB2 gene amplification and deletion of the 9p21 locus of the CDKN2A gene by the FISH method was determined and compared with the corresponding change in the expression of HER-2/neu and p16INK4A proteins. The first stage of the work included a retrospective study of archival material 41 cases of metastatic lesions of the lymph nodes of the neck by tumors of different histogenesis. Carrying out in these samples of MPM analysis of the size of tumor cell nuclei and comparing them with the size of normal lymphocytes allowed to objectively determine the categories of tumors by size: "really small cell tumors" with an area up to 30 μm2, perimeter up to 25 μm, but different values of roundness (the closer the latter was to unity, the rounder the nucleus ); "medium cell tumors" with an area of 30 - 50 μm2, perimeter 25 - 35 μm and also different values of the roundness, "large cell tumors" with an area of more than 50 μm2, perimeter - more than 35 μm and different values of the roundness; and separately identify a difficult to diagnose group of "small round blue cells tumors", which had ranges of 30 - 45 μm2 in area, 25 - 35 μm in perimeter and well-defined limits of roundness – 0.75 - 0.90. Given the anaplastic structure of tumors from the group of "small round blue cells" and the specificity of their IHC profile, it is advisable in case of obtaining such MPM indicators to change the standard primary IHC panel [Рan СК АЕ1/3, Vimentin, CD45, S100 and / or HMB45 to [Рan СК АЕ1/3, Desmin, CD45, CD56, CD99, S100 and / or HMB45] to reduce the time and cost of IHC study. Also, at the first stage of work from 41 cases of metastatic lesions of the lymph nodes of the neck with a clinical diagnosis of "malignant tumor of unknown origin" (category 1) after etrospective analysis of IHC, the percentages of CUP 2 and 3 categories were determined. After the primary panel of IHC, it was found that only a third - 34.15 % (14 of 41) were metastases of carcinoma, or socalled "previous CUP" (category 2). After the secondary panel of IHC, it was found that in 35.71 % (5 of 14) the expression of organ-specific markers was found (localization was determined), and the other 64.29 % (9 of 14) were divided into favorable phenotypes of the CUP syndrome, (21.43 % (3 of 14), 2 metastases of squamous cell carcinoma to the lymph nodes of the neck, 1 metastasis of Merkel cells carcinoma) and unfavorable phenotypes of the CUP syndrome (42.86 % (6 of 14); adenogenic low-grade cancers with phenotypes [Рan СК АЕ1/3 (+), Vіmentin (- or +), СК 7 (+)] and ndifferentiated cancers with [Рan СК АЕ1/3 (+), Vіmentin (- or +)]. In the absence of complete clinical data about the possible tumor origin, these 9 of 14 (64.28 %) metastatic cancers or 9 of 41 (21.95 %) of all neoplasm formed a group of "confirmed CUP" (category 3) and left patients actually undiagnosed. Checking the world statistics, where the 3rd category of "confirmed CUP" accounts for only 3-5 %, it is clear that the percentage of observations of metastatic lesions of category 3 in our work is significantly inflated, probably due to incomplete examination of cancer patients at the clinical "pre-biopsy stage". The second phase was devoted to the study of nine favorable phenotypes of CUP syndrome and the comparison of their characteristics with locally advanced forms of the corresponding carcinomas. In the third stage, from the 61 cases of CUP, IHC expression of p16INK4A was positive in 31 observations (50.8 %), which in the FISH study showed 24 (9 homozygous and 19 heterozygous) deletions of 9p21, which occurred in 8 of 9 (88.89 %) of the studied phenotypes of CUP, except for low-grade carcinoma with distribution along the midline of the body (extragonadal embryonic cell syndrome). IHC expression of HER-2/neu at the level of 2+/3+ was detected in 15 of 61 (24.59 %) CUP, but only in 9 observations had a real amplification of the ERBB2 gene on FISH in 5 of 9 (55.55 %) phenotypes: extragonadal embryonic cell syndrome, women with isolated papillary carcinomatosis, women with metastasis to the axillaries lymph nodes, metastasis of poorly differentiated NEC and adenocarcinoma with a profile of colorectal cancer. The small number of phenotypes with amplification of the ERBB2 gene on FISH makes it more specific for the differential diagnosis of CUP, compared with the deletion of 9p21 (p16INK4A / CDKN2A), which is probably universal in the acquisition of invasive and aggressive carcinomas of different localizations.