Vladimir M. Perelmuter

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.

Invasive and drug resistant expression profile of different morphological structures of breast tumors.

In order to understand invasive/adhesive and drug resistant properties of intratumor morphological heterogeneity of breast cancer, we compared the expression of genes responsible for the cell adhesion and for the drug resistance between distinct morphological structures of breast tumors. Tubular (hollow-like), alveolar (morula-like), trabecular, solid structures/patterns, and discrete (small) groups of tumor cells were isolated from invasive carcinoma of no special type (n=3) and invasive micropapillary carcinoma (n=1) of the breast using laser microdissection. The gene expression of cadherins, catenins, integrins, ABC transporters, GSTP1, and drug targets was analyzed using qRT-PCR. Expression of catenin genes was identified in almost all structures. In contrast, the expression of cadherin and integrin genes significantly varied depending on the morphological variant. Cadherin expression declined in the row: solid - alveolar and trabecular structures - discrete groups of tumor cells. Expression of integrins declined in the row: solid and alveolar - trabecular structures - discrete groups of tumor cells. For drug resistance genes, trabecular structures more often demonstrated activity of genes coding for ABC transporters compared to other morphological variants. These results indicate that intratumoral morphological heterogeneity in breast cancer correlates with expression profile of adhesion and drug resistance genes reflecting different patterns of invasive growth and responsiveness to chemotherapy.

Relationship between the expression of phosphorylated heat shock protein beta-1 with lymph node metastases of breast cancer

BACKGROUND Heat shock protein beta-1 (HspB1) is a chaperone of the sHsp (small heat shock protein). The common functions of sHsps are chaperone activity, inhibition of apoptosis, regulation of cell development, and cell differentiation, take part in signal transduction. OBJECTIVE To study the intracellular localization of phosphorylated features and non-phosphorylated forms of HspB1 in primary breast cancer cells and to evaluate their relationship with regional lymphatic metastasis. MATERIAL AND METHODS Tumor biopsies of breast tissue were collected from 100 patients with a confirmed diagnosis of invasive carcinoma, nonspecific type, between the ages of 31-80 years. Immunohistochemistry was used to determine the intracellular localization of phosphorylated and non-phosphorylated forms of HspB1. RESULTS The result of this study showed that biopsies from patients with lymph node metastasis exhibited significantly higher levels of phosphorylated forms of HspB1 in the nucleus and cytoplasm compared with the group without lymph node metastasis. Analysis showed that the expression of phosphorylated forms of the chaperone HspB1 correlates with the amount and percentage of lymph node metastases affected. CONCLUSION The nuclear expression of phosphorylated and non-phosphorylated forms of the chaperone HspB1 is a marker of tumor cells associated with lymphatic metastasis of breast cancer.

Relationship between morphological and cytogenetic heterogeneity in invasive micropapillary carcinoma of the breast: a report of one case

Invasive micropapillary carcinoma (IMPC) is a rare (up to 2%) and aggressive form of breast cancer.1 ,2 IMPC shows high intratumoral morphological diversity, which represents the degree of cell differentiation, as well as the architectural and invasive growth patterns of tumour cells. Morphologically, these tumours are characterised by the presence of hollow-like (tubular) and morula-like (alveolar) structures of cuboidal-to-columnar neoplastic cells, which are surrounded by empty spaces (retraction clefts) and display an inversion of cell polarity, detected by aberrant localisation of glycoprotein MUC-1 at the stromal–basal surface.1 ,3 In addition, micropapillary tumour clusters can be represented by tumour cells arranged in solid patterns (structures), trabecular structures and discrete (small) groups.3–6 It has been suggested that morphological diversity of IMPC is related to chemotherapy resistance,7 whereas the presence of retraction clefts around tumour clusters is associated with increased lymphangiogenesis and lymph node metastasis.8 Considerable intratumour morphological heterogeneity in breast cancer most likely results from genetic and epigenetic instability of the tumour cells.9 ,10 Previously, the relationships between morphologically distinct components and specific chromosome aberrations have been found in metaplastic and invasive ductal breast carcinomas,11 ,12 the latter is now classified as invasive carcinoma of no special type (IC NST), and is the most common histological type of breast cancer.1 IMPC demonstrates a heterogeneous pattern of chromosome aberrations, and tends to be genetically a more complex disease than IC NST.11 ,12 IMPC more often harboured gains of chromosomes 1q, 8q, 17q and 20q, and losses of 1p, 8p, 13q, 16q and 22q,13 ,14 which were emphasised by Marchio and coauthors13 as previously associated with breast tumours of high histological grade. In contrast, concurrent gain of 1q and 16p and deletion of 16q, related to low …

Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer

The biological properties of circulating tumor cells (CTCs), and their dynamics during neoadjuvant chemotherapy are important, both for disease progression prediction and therapeutic target determination, with the aim of preventing disease progression. The aim of our study was to estimate of different CTC subsets in breast cancer during the NACT (neoadjuvant chemotherapy). The prospective study includes 27 patients with invasive breast cancer, T2-4N0-3M0, aged 32 to 60 years. Venous heparinized blood samples, taken before and after biopsy, after each courses of chemotherapy (on days 3–7), and before surgical intervention, served as the material for this study. Different subsets of circulating tumor cells were determined on the basis of the expression of EpCAM, CD45, CD44, CD24, and N-Cadherin using flow cytometry. As the result of this study, it has been observed that significant changes in the quantity of the different subsets of circulating tumor cells in patients’ blood were observed after carrying out the 3rd course of NACT. NACT causes significant changes in the quantity of six CTC subsets, with various combinations of stemness and epithelial-mesenchymal transition (EMT) properties.

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.

Effect of small and radical surgical injury on the level of different populations of circulating tumor cells in the blood of breast cancer patients

Circulating tumor cells (CTCs) constitute a heterogeneous population. Some tumor cells are cancer stem cells (CSCs), while others are in the process of the epithelial-mesenchymal transition (EMT); however, most CTCs are neither stem cells nor in the EMT. This prospective study of 22 patients with nonspecific-type invasive carcinoma of the breast identified different populations of CTCs by flow cytometry in the blood of patients before biopsy, after biopsy and after surgical tumor removal without neoadjuvant chemotherapy. The results showed that minor surgical injury (biopsy) was accompanied by a significant increase in the blood levels of CTCs without signs of the EMT or stemness (Epcam+CD45-CD44-CD24-Ncadh-) and CTCs with signs of stemness and without signs of the EMT (Epcam+CD45-CD44+CD24-Ncadh-). Our results suggest that minor surgical injury to a tumor contributes to the release of CTCs into the bloodstream, including a population of stem cells.