Nadezhda V. Cherdyntseva

Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content

Introduction: Extracellular nucleic acids are found in human blood and cell culture medium as cell-free or being adsorbed at cell surface. In the last years, the circulating extracellular nucleic acids in blood were shown to be associated with certain diseases. Attempts are made to develop non-invasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Areas covered: This article reviews accumulating data regarding cell-free and cell-surface-bound extracellular nucleic acid nature and generation mechanisms. Their existence as a constituent of the naturally occurring complexes with proteins or membrane-bearing particles is discussed with regard to their homeostatic concentration and distribution in healthy donor blood which are significantly altered in cancer patients. Gene-target and whole-genome studies reveal significant differences in gene representation between extracellular DNA and genome DNA. Overrepresentation of regions with high transcription activity has led to proposal that extracellular DNA generation is strongly dependent on the parent genome functionality, which is associated with chromosome packaging and DNA methylation levels. Expert opinion: Recent studies provide evidence of the circulating nucleome organization complexity indicating that discovery of extracellular DNA generation and circulation patterns in healthy condition and cancer is essential to enable the development of proper approaches for the selection of valid diagnostic markers.

Intratumor heterogeneity: Nature and biological significance

Intratumor heterogeneity inherent in the majority of human cancers is a major obstacle for a highly efficient diagnosis and successful prognosis and treatment of these diseases. Being a result of clonal diversity within the same tumor, intratumor heterogeneity can be manifested in variability of genetic and epigenetic status, gene and protein expression, morphological structure, and other features of the tumor. It is most likely that the appearance of this diversity is a source for the adaptation of the tumor to changes in microenvironmental conditions and/or a tool for changing its malignant potential. In any case, both processes result in the appearance of cell clones with different undetermined sets of hallmarks. In this review, we describe the heterogeneity of molecular disorders in various human tumors and consider modern viewpoints of its development including genetic and non-genetic factors of heterogeneity origin and the role of cancer stem cells and clonal evolution. We also systematize data on the contribution of tumor diversity to progression of various tumors and the efficiency of their treatment. The main problems are indicated in the diagnosis and therapy of malignant tumors caused by intratumor heterogeneity and possible pathways for their solution. Moreover, we also suggest the key goals whose achievement promises to minimize the problem of intratumor heterogeneity and to identify new prognostic, predictive, and target markers for adequate and effective treatment of cancer.

Cell-Surface-Bound Circulating DNA as a Prognostic Factor in Lung Cancer

Since the mortality of lung cancer patients remains very high, development of prognostic methods essential for efficient therapy is an immediate task. This study was designed to assess the value of circulating DNA (cirDNA) in blood as a prognostic marker in patients with non–small cell lung cancer. The average concentration of cirDNA in plasma was shown to be similar in healthy donors and lung cancer patients. However, the concentration of cell‐surface‐bound circulating DNA (csb‐cirDNA) in lung cancer patients is significantly lower than that found in healthy donors (P < 0.0001) and correlates with a poor prognosis of tumor disease. Quantification of the cell‐surface‐bound DNA in blood of untreated patients allows persons with a poor prognosis of tumor disease to be detected with 94% sensitivity and 50% specificity.

Intratumoral morphological heterogeneity of breast cancer: neoadjuvant chemotherapy efficiency and multidrug resistance gene expression

In this study, the influence of intratumoral morphological heterogeneity of breast cancer on neoadjuvant chemotherapy (NAC) efficiency was investigated. In particular, we analysed the association of NAC response and pre- and post-NAC expression of the main multidrug resistance (MDR) genes - ABCB1, ABCC1, ABCC5, ABCG1, and ABCG2, with the presence of different morphological structures in breast tumors. In addition, the expression of MDR genes was investigated in different morphological structures and in their microenvironment by comparing probes obtained using laser microdissection. The results of this study showed that tumors with alveolar structures were more frequently NAC-nonresponsive than cases without this structural type (p = 0.0028, Bonferroni-corrected p = 0.014). The presence of trabecular structures in breast tumors was also associated with chemoresistance (p = 0.0272, Bonferroni-corrected p = 0.136). High expression of MDR genes was not found in alveolar structures (including their microenvironment) and in tumors containing this structural type. In contrast, more active MDR genes and expression of the ABCB1 gene were found only in trabecular structures. Taken together, our data indicate that breast tumors with alveolar structures possess resistance to NAC, which is not related to high expression of MDR genes, whereas chemoresistance of tumors with trabecular structures can depend on the expression level of ABCB1.

Crosstalk Between the FGFR2 and TP53 Genes in Breast Cancer: Data from an Association Study and Epistatic Interaction Analysis

To evaluate the potential for gene-gene interaction effects in sporadic breast cancer (BC) risk, we studied combinations of the fibroblast growth factor receptor 2 (FGFR2) rs1219648 and tumor protein 53 (TP53) rs1042522, rs1625895, and rs17878362 polymorphisms in BC patients (n=388) and healthy persons (n=275). In addition to a single-locus effect manifested by the association of FGFR2 rs1219648 and TP53 rs1042522 polymorphisms with high BC risk, depending on menopause status (0.001<p<0.05), we showed a highly significant cooperation between the examined polymorphisms in FGFR2 and TP53 in the determination of susceptibility to the disease. Indeed, we found that combinations of FGFR2 minor and TP53 major genotypes were associated with a high risk of BC, particularly in the postmenopausal period (0.01<p<0.05). In contrast, combinations of the FGFR2 and TP53 major genotypes had a protective effect against BC, especially in premenopausal women (0.001<p<0.01). Of note, all observations were estrogen receptor (ER) dependent. The significant crosstalk between FGFR2 and TP53 polymorphisms was also confirmed by multifactor dimensionality reduction and ordered combinatorial partitioning approaches (0.001<p<0.05). Taken together, data from the present study demonstrate the age- and ER-specific interplay between TP53 and FGFR2 in BC.

CD68+, but not stabilin-1+ tumor associated macrophages in gaps of ductal tumor structures negatively correlate with the lymphatic metastasis in human breast cancer

Tumor associated macrophages (TAM) support tumor growth and metastasis in several animal models of breast cancer, and TAM amount is predictive for efficient tumor growth and metastatic spread via blood circulation. However, limited information is available about intratumoral TAM heterogeneity and functional role of TAM subpopulations in tumor progression. The aim of our study was to examine correlation of TAM presence in various morphological segments of human breast cancer with clinical parameters. Thirty six female patients with nonspecific invasive breast cancer T1-4N0-3M0 were included in the study. Morphological examination was performed using Carl Zeiss Axio Lab.A1 and MiraxMidiZeiss. Immunohistochemical and immunofluorescence/confocal microcopy analysis was used to detect CD68 and stabilin-1 in 5 different tumor segments: (1) areas with soft fibrous stroma; (2) areas with coarse fibrous stroma; (3) areas of maximum stromal-and-parenchymal relationship; (4) parenchymal elements; (5) gaps of ductal tumor structures. The highest expression of CD68 was in areas with soft fibrous stroma or areas of maximum stromal-and-parenchymal relationship (79%). The lowest expression of CD68 was in areas with coarse fiber stroma (23%). Inverse correlation of tumor size and expression of CD68 in gaps of tubular tumor structures was found (R=-0.67; p=0.02). In case of the lymph node metastases the average score of CD68 expression in ductal gaps tumor structures was lower (1.4±0.5) compared to negative lymph nodes case (3.1±1.0; F=10.9; p=0.007). Confocal microscopy identified 3 phenotypes of TAM: CD68+/stabilin-1-; CD68+/stabilin-1+ (over 50%); and CD68-/stabilin-1+. However, expression of stabilin-1 did not correlate with lymph node metastasis. We concluded, that increased amount of CD68+TAM in gaps of ductal tumor structures is protective against metastatic spread in regional lymph nodes.

The presence of alveolar structures in invasive ductal NOS breast carcinoma is associated with lymph node metastasis

Dear Dr. Bedrossian: The morphological structure of the primary tumor is evidently related to clinical outcome, resulting from cancer progression clinically manifested as invasion, lymph node involvement, and distant metastases. The histological malignancy grading of operable breast cancer (BC) typically including tubular formation, mitotic counts, and nuclear atypia plays an important role in identifying patients with high risk of distant metastasis. High-grade breast tumors are known to be associated with the increased recurrence risk and hematogenic metastasis. Tumor invasion into blood vessels and perineural space, the high proliferative activity of cancer cells are also considered factors influencing poor outcome. As the most appropriate combination of factors contributing to risk of progression is still not completely clear due not only to lack of the relevant combination of factors considered but also to the heterogeneity within the tumor, it seems evident that the potential to use morphological prognostic criteria for unfavorable disease outcome is far from fully investigated. It is of particular concern in prognosis assessment for patients with invasive ductal breast carcinoma (IDBC), not otherwise specified (NOS) being the most common invasive BC. Actually, the majority of IDBC are presented by grade 2 tumors without invasion into blood vessels and perineural space. However, notwithstanding the same prognostic factors, patients with IDBC usually display the high disease outcome heterogeneity. Taken together, it has been supposed that primary IDBC exhibit morphological heterogeneity within the tumor, which could be associated with a different clinical prognosis. The aim of this work was to determine whether primary tumors of IDBC differ in morphological features, and how the morphological heterogeneity found within the tumor tissue might be associated with regional lymph node metastasis. The morphological heterogeneity in primary tumor and the status of regional lymph nodes were studied on routine hematoxylin and eosin sections in a consecutive series of 410 women with primary operable unicentric invasive breast carcinoma (T1-3N0-3M0). All individuals provided informed consent. Ethical permission was obtained from the ethical committee of Cancer Research Institute. None of patients had distant metastases before surgery, as evaluated by abdominal ultrasound, chest radiography (posteroanterior and lateral) and bone scans. Patients were aged more than 35 years, with a mean age of 44.5 6 4.3 for premenopausal and 59.1 6 7.8 for postmenopausal women. The basic characteristics of study groups of patients are shown in Table I. Of 410 patients, 308 received neoadjuvant chemotherapy (NAC) with the following regimens: cyclophosphamide, methotrexate, and five fluorouracil; cyclophosphamide, adriamycin, and five fluorouracil; taxotere and cytoxan; cyclophosphamide, adriamycin, and xeloda; and cyclophosphamide, methotrexate, and xeloda. The patients underwent radical mastectomy or radical breast resection. About 102 patients received no preoperative treatment. Surgery was followed by chemotherapy, radiotherapy, or hormonal therapy if indicated. Information from regular follow-up (range 5–10 years) was recorded on a database. All available slides (minimum four) were reviewed by two experienced pathologists. Histological type and grade were assessed according to WHO tumor classification. *Correspondence to: Evgeny Denisov, Ph.D., Department of Experimental Oncology, Cancer Research Institute, SB RAMS, Kooperativny Str. 5, Tomsk 634050 Russian Federation. E-mail: d_evgeniy@oncology.tomsk.ru Received 19 May 2011; Accepted 12 September 2011 DOI 10.1002/dc.21852 Published online 18 November 2011 in Wiley Online Library (wileyonlinelibrary.com).

Phenotypic Drift as a Cause for Intratumoral Morphological Heterogeneity of Invasive Ductal Breast Carcinoma Not Otherwise Specified

Abstract Invasive ductal carcinoma (IDC) not otherwise specified (NOS), the most common type of breast cancer, demonstrates great intratumoral morphological heterogeneity, which encompasses the presence of different types of morphological structures—tubular, trabecular, solid, and alveolar structures and discrete groups of tumor cells, the origins of which remain unclear at present. In this study of 162 IDC NOS patients, we investigated whether the distribution of different types of morphological structures is related to the basic clinicopathological parameters of IDC NOS. Our results showed that in patients with only one type of tumor structure, the presence of any one of the five types was equally probable; however, cases with two types of structures were more likely to contain trabecular structures than the other four types. The development of intratumoral morphological heterogeneity was not associated with menopausal status, tumor size, histological grade, hematogenic metastasis, or recurrence. However, the number of different types of morphological structures was significantly higher in luminal tumors than in triple-negative tumors. An increase in the frequency of lymph node metastasis correlated with the increased number of different types of structures in breast tumors; however, in contrast to premenopausal patients, this association was explained by the presence of alveolar structures in postmenopausal women. In addition, we showed a significant decrease in the numbers of positive lymph nodes in tumors with high numbers of morphological variants. The frequency of lymph node metastases and the number of positive nodes were generally independent features and formed by different mechanisms. Based on the evidence, the term “phenotypic drift” has been designated as the basis for the development of intratumoral morphological heterogeneity of IDC NOS.

Tumor-associated macrophages in human breast cancer parenchyma negatively correlate with lymphatic metastasis after neoadjuvant chemotherapy

Breast cancer is the leading cause of cancer death in women worldwide with high morbidity and mortality. Tumor-associated macrophages (TAM) are major innate immune cells in the tumor microenvironment controlling primary tumor growth and metastasis. Neoadjuvant chemotherapy (NACT) is a conventional pre-operative treatment for breast cancer. In the present study we examined the distribution of TAM in five distinct intratumoral morphological compartments of human breast cancer and their correlation with clinical parameters after NACT. Our data indicated that CD68+ but not stabilin-1+ TAM in areas with parenchymal elements negatively correlate with lymphatic metastasis after NACT. However, in cases where lymphatic metastases were detected (28 out of 50 analyzed samples) both amount of CD68+ and stabilin-1+ macrophages in the areas with coarse fibrous stroma directly correlated with the number of positive lymph nodes. In patients with complete response to the preoperative NACT the average score of CD68 expression in the areas with coarse fibrous stroma was lower compared with cases of a partial response and stable disease. We concluded that function of TAM after NACT depends on their intratumoral localization and local tumor microenvironment which plays an important role in polarization of macrophages towards tumor-suppressive or tumor-supportive types.

Plasma miR-19b and miR-183 as Potential Biomarkers of Lung Cancer

Lung cancer is a complex disease that often manifests at the point when treatment is not effective. Introduction of blood-based complementary diagnostics using molecular markers may enhance early detection of this disease and help reduce the burden of lung cancer. Here we evaluated the diagnostic potential of seven plasma miRNA biomarkers (miR-21, -19b, -126, -25, -205, -183, -125b) by quantitative reverse transcription PCR. Influence clinical and demographical characteristics, including age, tumor stage and cancer subtype on miRNA levels was investigated. Four miRNAs were significantly dysregulated (miR-19b, -21, -25, -183) in lung cancer patients. Combination of miR-19b and miR-183 provided detection of lung cancer with 94.7% sensitivity and 95.2% specificity (AUC = 0.990). Thus, miRNAs have shown the potential to discriminate histological subtypes of lung cancer and reliably distinguish lung cancer patients from healthy individuals.