Sergey V. Vtorushin

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.

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.

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.

Clinically relevant morphological structures in breast cancer represent transcriptionally distinct tumor cell populations with varied degrees of epithelial-mesenchymal transition and CD44 + CD24 - stemness

Intratumor morphological heterogeneity in breast cancer is represented by different morphological structures (tubular, alveolar, solid, trabecular, and discrete) and contributes to poor prognosis; however, the mechanisms involved remain unclear. In this study, we performed 3D imaging, laser microdissection-assisted array comparative genomic hybridization and gene expression microarray analysis of different morphological structures and examined their association with the standard immunohistochemistry scorings and CD44+CD24- cancer stem cells. We found that the intratumor morphological heterogeneity is not associated with chromosomal aberrations. By contrast, morphological structures were characterized by specific gene expression profiles and signaling pathways and significantly differed in progesterone receptor and Ki-67 expression. Most importantly, we observed significant differences between structures in the number of expressed genes of the epithelial and mesenchymal phenotypes and the association with cancer invasion pathways. Tubular (tube-shaped) and alveolar (spheroid-shaped) structures were transcriptionally similar and demonstrated co-expression of epithelial and mesenchymal markers. Solid (large shapeless) structures retained epithelial features but demonstrated an increase in mesenchymal traits and collective cell migration hallmarks. Mesenchymal genes and cancer invasion pathways, as well as Ki-67 expression, were enriched in trabecular (one/two rows of tumor cells) and discrete groups (single cells and/or arrangements of 2-5 cells). Surprisingly, the number of CD44+CD24- cells was found to be the lowest in discrete groups and the highest in alveolar and solid structures. Overall, our findings indicate the association of intratumor morphological heterogeneity in breast cancer with the epithelial-mesenchymal transition and CD44+CD24- stemness and the appeal of this heterogeneity as a model for the study of cancer invasion.Intratumor morphological heterogeneity in breast cancer is represented by different morphological structures (tubular, alveolar, solid, trabecular, and discrete) and contributes to poor prognosis; however, the mechanisms involved remain unclear. In this study, we performed 3D imaging, laser microdissection-assisted array comparative genomic hybridization and gene expression microarray analysis of different morphological structures and examined their association with the standard immunohistochemistry scorings and CD44+CD24- cancer stem cells. We found that the intratumor morphological heterogeneity is not associated with chromosomal aberrations. By contrast, morphological structures were characterized by specific gene expression profiles and signaling pathways and significantly differed in progesterone receptor and Ki-67 expression. Most importantly, we observed significant differences between structures in the number of expressed genes of the epithelial and mesenchymal phenotypes and the association with cancer invasion pathways. Tubular (tube-shaped) and alveolar (spheroid-shaped) structures were transcriptionally similar and demonstrated co-expression of epithelial and mesenchymal markers. Solid (large shapeless) structures retained epithelial features but demonstrated an increase in mesenchymal traits and collective cell migration hallmarks. Mesenchymal genes and cancer invasion pathways, as well as Ki-67 expression, were enriched in trabecular (one/two rows of tumor cells) and discrete groups (single cells and/or arrangements of 2-5 cells). Surprisingly, the number of CD44+CD24- cells was found to be the lowest in discrete groups and the highest in alveolar and solid structures. Overall, our findings indicate the association of intratumor morphological heterogeneity in breast cancer with the epithelial-mesenchymal transition and CD44+CD24- stemness and the appeal of this heterogeneity as a model for the study of cancer invasion.

Molecular biological predictors for kidney cancer

The paper considers the data available in the modern literature on studies of potential molecular predictors for renal cell carcinoma (RCC). Investigations of cell death markers, namely; Bcl-2 as an inhibitor of apoptosis, are of interest. Its high expression correlates with a more favorable prognosis. Inactivation of Berclin 1 that is an authophagy indicator in intact tissues gives rise to t high risk for tumorigenesis. At the same time, high Beclin 1 expression in the tissue of the tumor itself results in the lower efficiency of performed chemotherapy. Excess annexin A2 in the tumor promotes the growth and invasion of cancer cells. Patients with tumor over-expression of SAM68 protein involved in cell proliferation have a lower overall survival rate. The lifespan of patients without distinct metastases survive significantly longer in the overexpression of epithelial cell adhesion molecule (EpCAM). High PD-L1 protein expression on the cell membrane is considered to be a potential marker of effective immunotherapy for RCC.

A LINK BETWEEN AUTOPHAGY REGULATORY PROTEINS M-TOR AND BECLIN-1 AND PARAMETERS OF LYMPHOGENIC METASTASIS IN COLORECTAL CANCER

Currently the impact of autophagy on carcinogenesis remains understudied. on the one hand, autophagy acts as a tumor suppressor, as it activates degradation of oncoproteins, toxic proteins, and damaged cell organelles, that may be aggressive and lead to dNA damage. on the other hand, autophagy may promote tumor cell survival under hypoxia and in the presence of reactive oxygen species, which occurs primarily due to blocking of apoptosis mechanisms, raising the chances for maintaining tumor clone dynamics. Autophagy regulation is a complicated and multi-stage process. The main regulator here is a signaling pathway that activates serine/threonine protein kinase m-ToR (the mammalian target of rapamycin). data on the impact of autophagic proteins ATG5, LC3A, LC3B, and Beclin-1 on malignant cell survival as well as on tumor growth and progression have been reported in literature. However, studies aimed at seeking possible relationships between autophagy and pathogenetic mechanisms of carcinogenesis are of great interest. The aim of the study is to investigate a relationship between the expression parameters of autophagy regulatory proteins m-ToR and Beclin-1 and the features of lymphogenic metastasis in colorectal cancer. Materials and methods. The study included 105 patients with T1-4N0-3M0 colorectal cancer treated in the Thoracic and Abdominal department of Cancer Research Institute of Tomsk Research Medical Center from 2012 to 2015. The average age of patients was 59.7±4.3 years. Morphological verification of the diagnosis was performed on the biopsy samples of primary tumor tissue. Staging of colorectal cancer was determined according to the TNM classification of malignant tumors (2002). Results. Analysis of the frequency of lymphogenic metastasis depending on the presence or absence of m-Tor and Beclin-1 expression in tumor cell cytoplasm revealed a statistically significant link between these variables. Conclusion. The obtained findings clearly exhibit that deceleration or loss of autophagic activity in the tumor is accompanied by implementation of lymphogenic dissemination, which is a predictor of an unfavorable outcome of the disease.

ASSOCIATION BETWEEN SPECIFIC FEATURES OF GATA3 TRANSCRIPTION FACTOR EXPRESSION AND LYMPH NODE METASTASIS IN LUMINAL BREAST CANCER

Currently, the study of the markers of cell differentiation, proliferative regulators, and molecules involved in the development of drug resistance mechanisms in breast cancer is extremely important. The transcription factor GATA3 plays an essential role in the differentiation and proliferative activity of luminal breast cancer cells, being a tumor suppressor. The GATA3 positive expression is most frequently observed in invasive carcinoma of no special type. High expression of GATA3 is associated with low-grade ER-positive cancer with a favorable prognosis. Low GATA3 expression is observed in patients with high-grade and hormone receptor-negative cancer. The study of GATA3 expression is necessary for understanding the development of drug resistance mechanisms and developing approaches to overcome them as well as for determining the response to hormone therapy. Aim. The present study was undertaken to study the expression characteristics of the transcription factor GATA3 in patients with luminal breast cancer and to evaluate their relationship with the parameters of lymphogenous metastasis. Material and methods. The study included 64 patients with stage T1–4N1–3M0 invasive breast cancer. The primary tumor tissue and all removed lymph nodes were morphologically examined. The diagnosis was established according to the WHO criteria (2012). Results. Low GATA3 expression was associated with a high risk of lymph node metastases, while high GATA3 expression was associated with the absence of lymph node metastases. Heterogeneous GATA3 expression was associated with high risk of lymph node metastasis, and as a consequence, with poor prognosis. Conclusion. The relationship between the expression of GATA3 protein and lymphogenic metastasis in patients with luminal breast cancer was found.

Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model

Background European liver fluke Opisthorchis felineus, causing opisthorchiasis disease, is widespread in Russia, Ukraine, Kazakhstan and sporadically detected in the EU countries. O. felineus infection leads to hepatobiliary pathological changes, cholangitis, fibrosis and, in severe cases, malignant transformation of bile ducts. Due to absence of specific symptoms, the infection is frequently neglected for a long period. The association of opisthorchiasis with almost incurable bile duct cancer and rising international migration of people that increases the risk of the parasitic etiology of liver fibrosis in non-endemic regions determine high demand for development of approaches to opisthorchiasis detection. Methodology/Principal findings In vivo magnetic resonance imaging and spectroscopy (MRI and MRS) were applied for differential assessment of hepatic abnormalities induced by O. felineus in an experimental animal model. Correlations of the MR-findings with the histological data as well as the data of the biochemical analysis of liver tissue were found. MRI provides valuable information about the severity of liver impairments induced by opisthorchiasis. An MR image of O. felineus infected liver has a characteristic pattern that differs from that of closely related liver fluke infections. 1H and 31P MRS in combination with biochemical analysis data showed that O. felineus infection disturbed hepatic metabolism of the host, which was accompanied by cholesterol accumulation in the liver. Conclusions A non-invasive approach based on the magnetic resonance technique is very advantageous and may be successfully used not only for diagnosing and evaluating liver damage induced by O. felineus, but also for investigating metabolic changes arising in the infected organ. Since damages induced by the liver fluke take place in different liver lobes, MRI has the potential to overcome liver biopsy sampling variability that limits predictive validity of biopsy analysis for staging liver fluke-induced fibrosis.

Abstract A26: The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance

Background. The crosstalk between estrogen receptor alpha (ERα) and growth factor receptors is an essential part of tamoxifen resistance and the vascular endothelial growth factor/ tyrosine kinase domain receptor (VEGF/KDR) signaling pathway is clinically significant in the mechanisms of this resistance. The aim of the present study was to analyze the association of distribution pattern of ERα expression, VEGFR2 expression level, ESR1 and KDR single nucleotide polymorphisms (SNPs) with tamoxifen response in hormone receptor-positive primary breast cancer. Material and methods. Formalin-fixed paraffin-embedded and fresh frozen breast cancer tissue of the primary tumor was obtained from 97 hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen at the Tomsk Cancer Research Institute. The distribution patterns of ERα expression and Ki-67 protein expression were determined by immunohistochemistry. The quantitative expression levels of VEGFR2 (CD309) and p-Akt473 was measured using a flow cytometry. TaqMan SNP assays were used for genotyping ESR1+30T>C (rs2077647), ESR1 2014G>A (rs2228480), KDR-604T>C (rs2071559) and KDR 1192G>A (rs2305948) polymorphisms. Progression-free survival (PFS) was used as end-point for survival analyses. Results. Patients who did not benefit from tamoxifen treatment (tamoxifen resistance group -TR) showed significantly higher heterogeneous ERα expression than tamoxifen sensitive patients (TS; 81.1% and 58.3% respectively, P = 0.021). We found that ESR12014A mutant allele carriers were more prevalent in TR patients than in TS group (26.3% vs. 8.0%, respectively, P = 0.009). Similarly, the KDR -604T allele of rs2071559 was statistically significant related with tamoxifen resistance (P = 0.034). However, the KDR 1192G allele of rs2305948 were significantly less common in the tamoxifen progressive group compared to the tamoxifen sensitive patients (38.0% vs. 90.0%, respectively, P = 0.035). In addition, the variant homozygote KDR 1192GG (vs. AA) of rs2305948 was associated with VEGFR2 expression (r = -0.524, p = 0.012). VEGFR2 expression levels also showed borderline significant correlation with p-Akt473 expression (r = 0.368, p = 0.070). The Kaplan-Meier survival analysis demonstrated a significant relationship of heterogeneous distribution of ERα expression with poor survival of tamoxifen treated patients (log-rank P = 0.009). Moreover, the PFS for the patients with the KDR -604TT genotypes of rs2071559 were worse than for the patients with the CC genotype (log-rank P = 0.123). Conclusion. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation can serve as potential predictors for hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen and thus provide more information for optimizing patient9s selection to tamoxifen adjuvant therapy. Citation Format: Nataliya Babyshkina, Sergey Vtorushin, Stanislav Patalyak, Tatyana Dronova, Elena Slonimskaya, Nadejda Cherdyntseva. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A26.