Evgeny V. Denisov

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.

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.

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.

Association between TP53 gene ARG72PRO polymorphism and chromosome aberrations in human cancers.

It is well known that the TP53 gene considerably influences on DNA repair processes. Polymorphisms in the TP53 gene, particularly the well‐known Arg72Pro in codon 72 of exon 4 (Ex4+119 G>C; rs1042522), can modify the functionality of the p53 protein and activation of DNA repair. Actually, polymorphic variants Arg and Pro were found to have different properties of regulation of TP53‐dependent DNA repair target genes, that can effect various levels of chromosome aberrations in cancer patients with these genotypes. Here, we studied frequency of chromatid breaks (CB), chromosome‐type aberrations (CTA) and aberrant cells (AC) in cancer patients (n = 102) with various Arg72Pro genotypes. It was shown that the Arg variant of TP53 gene is associated with high frequency of AC and chromatid breaks. That is Arg/Arg carriers have more different chromosome aberrations in comparison to individuals with Arg/Pro and Pro/Pro genotypes (P < 0.05). Conversely, the lowest level of AC and chromatid breaks were detected in cancer patients with the Pro/Pro genotype. A completely unexpected result was that women with Arg/Arg genotype had the most high frequency of CB and AC in comparison to Arg/Pro and Pro/Pro women carriers (P < 0.001). In the group of male patients we did not show any differences in chromosome aberrations between carriers of Arg72Pro genotypes. In conclusion, the TP53 gene Arg72Pro polymorphism appreciably influence on occurrence of chromosome aberrations in cancer. Mol. Carcinog.

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.

Coordination of TP53 abnormalities in breast cancer: data from analysis of TP53 polymorphisms, loss of heterozygosity, methylation, and mutations.

AIMS We have studied whether TP53 rs1042522, rs17878362, and rs1625895 alleles having a protective effect against breast cancer (BC) will be lost in tumors, whereas those allowing disease development will be retained. METHODS Analysis of TP53 polymorphisms was performed in blood leukocytes and tumors from 80 Caucasian BC patients. In addition, TP53 loss of heterozygosity (LOH), methylation, and mutations were studied in tumor DNA of BC individuals with loss of alleles of TP53 polymorphisms. RESULTS In breast tumors of patients heterozygous for TP53 polymorphisms, we detected loss of rs1042522 C and G and rs17878362 A2 alleles; however, the loss of the C allele was preferential. We found that loss of TP53 alleles, namely rs1042522 C, has been caused by an LOH mechanism, namely TP53 deletions, whereas TP53 point mutations frequently occurred in the retained G allele (p=0.03). In addition, we showed that BC patients with rs1042522 CC genotype were characterized by only unifocal tumors and decreased frequency of lymph node metastases (p=0.03). CONCLUSIONS Taken together, we showed the preferential loss of the rs1042522 C allele, which is protective against BC progression, in breast tumors. Also, the loss of the C allele, which encodes p53 protein with the best DNA repair capability according to literature data, may create prerequisites for mutations, but not for methylation in a retained G variant, as we found here. However, these results need to be confirmed because of the limited statistical power of the present study and the small sampling.

TP53 Gene Polymorphisms in Cancer Risk: The Modulating Effect of Ageing, Ethnicity and TP53 Somatic Abnormalities

Evgeny V. Denisov1, Nadezhda V. Cherdyntseva1, Nicolay V. Litviakov1, Elena A. Malinovskaya1, Natalya N. Babyshkina1, Valentina A. Belyavskaya2 and Mikhail I. Voevoda3 1Cancer Research Institute, Siberian Branch of Russian Academy of Medical Sciences, Tomsk, 2Research Center of Virology and Biotechnology VECTOR, Koltsovo, 3Institute of Internal Medicine, Siberian Branch of Russian Academy of Medical Sciences, Novosibirsk, Russian Federation

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 …