Stromal Changes in Colon Blastomogenesis Associated with Development of Hypoxia in the Foci of Dysplasia.

We studied the features of reaction of the colon stromal cells (lymphohistiocytic population, fibroblasts, and blood vessels) to the appearance and progression of dysplasia in the colon epithelium against the background of increasing ischemia in the colon mucosa. The morphological material from 92 patients treated for benign processes and colon cancer in 2002-2016 was examined. Common histological methods and a complex immunohistochemical staining were used. The stromal cells of the colon mucosa, mainly lymphohistiocytic cells, undergo certain quantitative changes specific for each type of cells during progression of dysplasia and aggravation of ischemia in the mucosa. Some cells, e.g. plasma cells, presumably contribute to tissue hypoxia in the stroma. Most stromal cells, except interdigitating S100+ dendritic cells and CD10+ fibroblasts, decreased at the stage of grave dysplasia and cancer in situ. Low effectiveness of the immune defense can be partly explained by impairment of the function of stromal cells as a result of hypoxia in the microenvironment.

Different Patterns of Vascularization in Preinvasive States and at the Initial Stages of Invasive Growth of the Neoplasms.

Tissue samples obtained during surgery from 90 patients with malignant neoplasms of various localizations were studied (the presence of precancerous dysplastic alterations and their transition to invasive cancer in the slides were obligatory condition). In addition to traditional histological methods, immunohistochemical reactions for detection of HIF-1α, GLUT1, CAIX, and CD31 were performed. At the precancerous stage including cancer in situ, progressive signs of reduced blood vessel density and hypoxia were observed. At the earliest stages of invasion, hypoxia was compensated by abundant vascularization of the stroma, which was confirmed by disappearance of hypoxia markers in tumor cells and their persistence in the deep layers of the tumor far from blood vessels. At the same time, the ischemic phenotype was preserved in tumor cells even in abundantly vascularized stroma, which can attest to deep metabolic changes in some tumor cells similar to the Warburg effect. Thus, the initial stages of carcinogenesis are associated with reduction of the vascular network up to the complete absence of blood vessels in the cancer in situ. After migration to the vascularized subepithelial stroma, e.g., having started invasion that compensated for hypoxia, the tumor cells no longer expressed markers of hypoxia, except the cells located far from blood vessels. In parallel, neoplastic cell clones that presumably have changed their phenotype and transformed their metabolism similar to the Warburg effect were detected. In the deep layers of the tumors, these cells coexist in different proportions. Analysis of the content of these cells, their alternation, and mutual transformation will be very valuable for estimating the sensitivity of tumor cells to therapeutic measures.

The Problem of Vascularization of Precancerous Lesions of the Colon and Development of Hypoxia at the Incipient Stages of Blastomogenesis.

The features of the blood supply in the colon mucosa during the development of hyperplastic and dysplastic processes, and early neoplastic lesions were studied. The study cohort comprised the material of 92 patients of the N. N. Blokhin National Medical Research Center of Oncology collected during 2002-2016. The initial stages of hyperplastic, atypical processes developed against the background of enhanced angiogenesis. During the development of adenomatosis with concomitant dysplasia, the blood supply of newly formed structures decreased, which was associated with overexpression of markers of tissue hypoxia Hif1, CAIX, and Glut1. The expression of these markers was maximal in the foci of highly pronounced dysplasia, but was not detected in the foci of carcinoma in situ and microinvasion, when the cells reach the stroma with abundant vascular supply. It can be hypothesized that hypoxia forces the cells to search the territories with sufficient vascularization and migrate to other structures, which finally manifests in invasion and destruction of adjacent tissues and dissemination of these cells in the body (formation of metastasis).

Undifferentiated sarcoma of the pericardium after radiation therapy for Hodgkin's lymphoma.

Primary sarcomas of the pericardium are extremely rare malignant tumors of the heart. The incidence of sarcoma increases after radiation therapy in the field of breast. The specific features of this case report are the difficulties in diagnostics of undifferentiated spindle-cell sarcoma of the pericardium and the connection between the disease and the radiation therapy for Hodgkin's lymphoma.

Intraepithelial ischemia is a principal factor promoting cancerization of the covering epithelial tissues.

Prominent angiogenesis, which is a hallmark of invasive cancer is preceded at the precancerous stage by marked ischemia. Our hypothesis proposes a structural mechanism responsible for altering blood flow in the covering epithelium and leading to marked reduction of vascularization in the foci of dysplasia. This mechanism varies from one type of epithelium to another. In squamous epithelium only basal cells are in direct contact with stromal vessels. To supply nutrients to the rest of the cells located at different levels, the subjacent stroma forms excrescences which penetrate upward together with blood capillaries. As soon as precancerous dysplastic alterations start and progress the number of intraepithelial blood vessels simultaneously decreases, thus leading to ischemia which precedes or promotes malignization of the covering squamous epithelium. To compensate for the deficit in blood supply, the dysplastic cells penetrate deeper into the underlying stroma, commencing invasion. Thus, the cells destroy the subjacent stroma not because they are initially "malignant", but due to ischemia which provokes the search for nutrients. Comparing squamous epithelium with glandular respiratory epithelium shows that the latter contains no blood capillaries at all. However, unlike squamous epithelial coverings, in respiratory epithelial covering, each cell is attached directly to the basal membrane and has ample access to the blood supply. Covering respiratory epithelium itself seldom gives rise directly to malignant growth. Cancerization of this type of epithelium occurs in the foci of squamous metaplasia. The latter are not supplied by a sufficient amount of blood vessels and in the majority of cases remain fragile and vulnerable structures, easily prone to malignization. Further study of these phenomenon should include the clarification of the influence of carcinogenic agents on the mechanism of adequate vascularization at the precancerous stage.

[Ovarian germ cell tumors in girls].

Morphological structure of tumor influences on the clinical course of the disease in children with germ cell tumors. Patients with ovarian dysgerminoma at the time of diagnosis are significantly older than patients with immature teratoma and yolk sac tumor. Immature teratoma and mixed germ cell tumors are significantly larger compared to other germ cell tumors. Yolk sac tumor and embryonal carcinoma are the most common cause of emergency surgical interventions and are accompanied by rupture of tumor capsule.

Impairment of vascularization of the surface covering epithelium induces ischemia and promotes malignization: a new hypothesis of a possible mechanism of cancer pathogenesis.

PURPOSE: To study the peculiarities of vascularization at the stromal-epithelial interface in different types of epithelia and their alterations in precancerous lesions. MATERIALS AND METHODS: Peritumoral tissues of 310 patients, tissues of 180 healthy persons and of 50 human embryos and fetuses were used. Traditional histological as well as immunohistochemical methods have been used. RESULTS: The study reveals that the occurrence of blood capillaries in surface squamous epithelium is an ordinary event, both in healthy persons and in peritumoral regions of the patients with squamous cell carcinoma. Glandular epithelial coverings, as well as transitional epithelium, do not contain blood vessels. In squamous epithelium, only basal cells are in contact with the membrane and underlying stroma, the cells of the upper layer receiving nutrients through diffusion. Thus, the cells of squamous epithelium are more vulnerable to blood deficiency, since for instance in the pseudo-multilayered respiratory epithelium each cell is attached directly to the basal membrane and has more ample access to the blood supply. Metaplastic squamous epithelium has a markedly reduced vascularization and seems to be more sensitive to carcinogenic stimuli. High-grade dysplastic squamous epithelium and carcinoma in situ do not contain blood vessels. CONCLUSION: The process of redistribution of vascular network occurring at the interface of epithelial-stromal frontier plays an important role in maintaining the adequate metabolism of cells including those of epithelial covering. Impairment of this mechanism most probably promotes precancerous alterations.

[Mutation scanning of short amplicons: optimization of DNA melting conditions].

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.

[The problems of yolk sac tumor morphogenesis in a light of the tumor stem cell theory].

The analysis of possible morphogenesis of the different structures in human yolk sac tumor has been considered. The author has supposed that features of blood vessel microarchitecture formation and perpetual differentiation of tumor cells or theirs functional modification play a crucial role in the morphogenesis of YST. The immunohistochemical investigation of some stem cells markers has showed the necessity of accounting of their distribution pattern in various cellular structures for the differential diagnosis of morphogenetical steps of YST. The growth of tumor cells differentiation rate correlates with increasing of stem cells markers expression as well c-kit > OCT4 > CD30 > PLAP.

[New feasibility of using a tissue culture technique in diagnostic oncomorphology in case of neuroblastoma and breast cancer].

The paper gives the results of investigating the cultured tissues from patients with breast cancer (BC) and those with neuroblastoma, by applying both the traditional studies and molecular genetic techniques (FISH). The cultures from the patients with neuroblastoma represented as three cell types--N, S, and I. There are certain similarities between the cell composition in the culture medium and the histological structure of the tumor. Mature neoplasms of the ganglioneuroma type contain S-type cells only while intermediate type I cells are a predominant element of immature neuroblastomas. The presence of a considerable number of I cells in the explants appears to suggest a poor prognosis even when the tumor has a comparatively mature structure as a whole. The results of FISH on explant cells presented a means of detecting a broad range of points that are not recorded on histologic specimens--amplification on the metaphase plate, localization of an amplified product, amplification on one chromosome, and double acentric chromosomes. The results of cultivation of BC cells are difficult to systematize although they offer the advantage of performing the FISH reaction. The authors recommend that the tissue culture method should be more extensively used in the day-to-day diagnostic work of pathology laboratories.

[Molecular genetic study of benign pigmented neoplasms by fluorescence in situ hybridization].

Twenty samples of benign pigmented neoplasms of the skin, including 9 intradermal nevi and 11 complex ones, were investigated. Fluorescence in situ hybridization was used to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes. Analysis of the findings revealed no significant changes characteristic of melanoma in the nevi. However, the authors established that there was a direct correlation between the copy number of the MYB and RREB1 genes and that the amount of the MYB gene most frequently deviated from the normal values. In addition, a relationship was found between the number of MYB gene copies and the depth of the epidermal layer. In cases of an intradermal nevus, the copy numbers of the CCND1 and MYB genes were shown to vary more greatly than in cases of a complex nevus.

[The morphology of triple negative breast cancer].

Triple negative breast cancer (TNBC) does not express estrogen and progesterone receptors and amplified Her2/neu gene. Examining 90 cases of TNBC by traditional, histological, and immunohistochemical studies (70 patients) has indicated the heterogeneity of this group of breast neoplasms. It is represented by a great variety of the histological types of mainly high-grade breast cancer. The markers of basaloid differentiation (cytokeratins 14, 5/6, 17, and p63) in the tumor cells were revealed in various ratios in 49 patients. The above markers were absent in 21 cases. Poor prognosis in TNBC seems to be largely associated with the presence of the basal-like phenotype rather than that of the triple negative phenotype. Much work to determine specific quantitative parameters is to be done to define the nosological specificity of mainly the basal-like subtype of TNBC.

[PTEN gene in triple-negative breast cancer].

The paper gives the results of studying the expression of the PTEN gene product by an immunohistochemical method, as well as deletion of the gene by fluorescence in situ hybridization in the tumor cells of 80 patients with triple-negative breast cancer (TNBC). The gene product was absent in the tumor cell nuclei in 56 products. The bulk of the remaining patients who showed a positive response had lobular carcinomas or tumors with an unidentified microscopic variant due to secondary posttherapeutic changes. Lobular carcinomas in a control group presented by patients without TNBC also contained the PTEN gene product. Therefore, all positively responding tumor cells failed to express basaloid markers and androgen receptors. The immunohistochemically detectable absence of the PTEN gene product is usually coupled with deletion at locus 10q23; however, in several cases the negative immunohistochemical reaction is associated with no deletion at the above locus, which suggests that there are mechanisms for PTEN gene dysregulation other than deletion in TNBC. The presence of the PTEN gene product in the tumor cells is associated with good prognosis in patients with TNBC.

[Ectopic hormone production of beta-subunit of human chorionic gonadotropin by cells of signet ring cell carcinoma of the stomach].

The ectopic hormone production by tumor cells that have a light optical structure of typical non-endocrine cancers has long attracted the attention of investigation all over the world. Specifically, this concerns the phenomenon of the ectopic production of the beta-subunit of human chorionic gonadotropin (beta-hCG) by non-germinogenic tumors, which is, according to R.K. Iles's data, encountered in 20-40% of malignant epithelial tumors. Despite the fact that beta-hCG synthesis is traditionally regarded as the prerogative of germinogenic tumors, the world's literature contains strong evidence for latent beta-hCG gene expression and many cases of clinically evident manifestation of the effects of chorionic gonadotropin. The latter fact, if a clinician and a pathologist are unaware of the behavior of a tumor, may give rise to a diagnostic error and incorrect treatment policy.

[Some fundamental notions of oncomorphology in the light of modern molecular biology advances].

Introduction of molecular biological studies into oncomorphology has made investigators reconsider many fundamental notions of the histogenesis, morphogenesis, and microscopic structure of human neoplasms. Tumor cell differentiation is a more dynamic process, a less fixed concept; hence it is necessary to do away with the rigid frameworks of cancer nosological entities and with a number of postulates on tumor histogenesis, including the rudiments of blastemic tissue changed during embryogenesis. The morphogenesis of a tumor has proven to be frequently determined by the variants of the specific translocations that may, in addition to its miscroscopic structure, affect a great variety of the clinical manifestations of disease. The molecular portrait of a tumor, the result of gene expression peculiarities can substitute for traditional cancer nosological entities although new classifications should be based on advances in oncomorphology.

[HERV-K-associated carcinogenesis: co-expression of viral and cellular proteins in the development of human germ-cell tumors].

To elucidate the role of some viral and cellular proteins in the occurrence and development of HERV-K-associated germ-cell tumors (GCT), reverse-transcription polymerase chain reaction using specific primers has been employed to study the transcription of the protein Rec HERV-K and the possible interaction of the protein Rec(cORF), that has transforming properties, and the cellular protein PLZF, that is a negative regulator of cell division, in human GCT tissues, in the testicular parenchyma adjacent to a tumor, and in the normal testicular tissues. It was shown that there was expression of Rec(cORF) of mRNA, rather than cellular PLZF in all malignant GCT tissues, this led to the conclusion that no interaction occured between the Rec HERV-K and PLZF proteins in the GCT cells. At the same time co-expression of Rec and PLZF protein was first revealed at the level of transcription in the testicular parenchyma adjacent to a tumor that exhibited carcinoma in situ cells. By taking into account that the protein Rec HERV-K has transforming activity and it is presumed to be Implicated in the development of GCT, the authors discuss a possible role in the Rec HERV-K/HTDV and cellular PLZF interaction in the pathogenesis of GST at the early stages of its genesis.

[Surgery for mesenchymal tumours of the mediastinum].

Results of treatment of 52 patients with mesenchymal tumours of the mediastinum. Clinical finding is various and nonspecific. Computer tomography and MRT are the most informative diagnostic techniques. Among 52 patients with mesenchymal tumours of the mediastinum, 40 patients were treated surgically and 12 patients got conservative treatment. Chemotherapy and radiotherapy were carried out in 7 patients in pre- and postoperative periods. Radical surgical treatment was carried out in 21 patients with benign tumors. Among 19 patients with malignant tumors, 42% of patients got radical surgical treatment, and 58 % of patients got palliative therapy in combination with chemotherapy. Benign tumors have a favorable prognosis after surgical treatment. Major factors of the prognosis of surgical treatment are dissemination and the histologic form of a tumor as well as type of surgical intervention. Re-operation is necessary in case of relapse.

Immunophenotypic profile of biomarkers related to anti-apoptotic and neural development pathways in the Ewing's family of tumors (EFT) and their therapeutic implications.

BACKGROUND: Ewing's family of tumors (EFT) comprises a broad spectrum of tumors composed of primitive committed cells with neuroectodermal capacity. The degree of neural differentiation within EFT, as measured with morphological features and expression of neural markers, delimits two members: Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET). Molecules such as c-kit and its ligand (Stem cell factor, SCF), CD95 (FAS), CD95L (FASL), IGF-IR, protect EFT cells from apoptosis, whereas c-erb-B2, erythropoietin (EPO) and its receptor (EPO-R) participate in the maturation of primitive committed neuroectodermal cells and in the normal embryonal brain development. The aim of the present study was to analyse the expression of these molecules in paraffin-embedded material from a series of EFT. MATERIALS AND METHODS: Forty-five cases of EFT (23 typical ES, 4 atypical and 18 pPNET) were analysed following the immunohistochemical LSAB method, with antigen retrieval heating using an autoclave, citrate buffer pH 6.0 and the following primary antibodies: FAS (APO-CD 95), FAS-L, c-kit, SCF, IGF-IR and c-erbB2. The expression was evaluated independently by three of the authors and the final score (0 to 3+) was based on the intensity and percentage of positively stained cells. In a second cooperative analysis, tissues from 30 cases of EFT (15 typical, 3 atypical and 12 PNET) were immunostained with EPO and EPO-R. RESULTS: High expression of c-kit/SCF (2+, 3+) was detected in 28/45 cases of EFT (62.2%), whereas FAS-FAS-L and IGF-IR were observed in 16/45 (37.7%) and 9/45 (20%), respectively. Regarding the neuroectodermal pathway, membranous and cytoplasmic expression of c-erb-B2 was observed in 9/45 (20%) EFT, regardless of the morphological and immunohistochemical expression of conventional neural markers. High expression of EPO and EPO-R was observed in 20/30 EFT (66.6%). CONCLUSION: C-kit/SCF and EPO/EPO-R seem to participate in the pathway of anti-apoptotic and proliferative advantage, while c-erb-B2 does not play an important role in the neuroectodermal differentiation pathway in EFT cells.

Downregulation of genes encoding for subunits of adaptor complex-3 in cervical carcinomas.

We explored the expression of four genes encoding for subunits of AP-3 in cervical tumors and cancer cell lines. Using RT-PCR we demonstrated more than twofold decrease in the levels of mRNA of AP3D1, AP3B1, AP3M1, and AP3S1 in 32, 28, 23, and 26% tumors in comparison with normal tissues of uterine cervix, respectively. The level of mRNA of at least one subunit was decreased in 28 out of 47 (60%) of tumors and in four out of five cancer cell lines in comparison to tissues adjacent to tumors. The suppression of expression of any of the subunits was revealed in 15 out of 28 cases (54%). The expression of two and more subunits was decreased simultaneously in different combinations in 13 cases (46%). This fact testifies to the lack of a common mechanism of downregulation of four subunits in tumors. There is a tendency to more frequent suppression of AP-3A expression in tumors associated with lymphatic node metastases as compared with tumors without metastases (P = 0.034). Thus, here we demonstrate for the first time the decrease in expression of genes encoding for AP-3A subunits in tumors.

[Antibodies to stuctural proteins of endogenous retrovirus of the HERV-K/HTDV family as markers of human germ cell tumors].

Human germ cell tumors (GCT) have been found to be closely associated with the expression of HERV-K/HTDV proviruses and most patients with GCT produce antibodies to the major HERV-K/HTDV Gag and Env proteins. The findings have shown a strong association of the level of HERV-K/HTDV antibodies with the clinical course of the disease and therapy success, which makes it possible to confirm the fact that viral protein antibodies may be used as an additional marker of GCT.