N. Litviakov

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.

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.

Phenomenon of loss of heterozygosity in tumour tissue of breast cancer: association with expression of monoresistance genes

Purpose of work. To perform a genome-wide association study of loss of heterozygosity (LOH) with monoresistance genes expression during neoadjuvant chemotherapy (NAC) in breast cancer.Materials and methods. The study involved 68 patients with breast cancer. The tumour stages were IIAIIIB. RNA was extracted from tissue specimens (before and after NAC) using RNeasy Plus mini Kit (Qiagen, Germany). Expression profiling of the RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, BRCA1 genes was carried out using quantitative real-time PCR (qPCR). DNA was extracted from 68 biopsy specimens of tumour tissues using QIAamp DNA mini Kit (Qiagen, Germany). LOH status was detected using microarray analysis using high density DNA-chip manufactured by Affymetrix CytoScanTM HD Array company.Results. As a result of the study of loss of heterozygosity was evaluated in 13815 genes. The frequency of LOH varied from 0% to 63%. The highest incidence of heterozygosity loss events is characteristic for genes of 16, 17 and the X-chromosome. Our study established that the phenomenon of loss of heterozygosity in monoresistance genes (BRCA1, ERCC1, RRM1, TOP1, TOP2A, TUBB3 and TYMS), is not associated with their level of expression in the tumor. A statistical association has been found between LOH and level of expression of the studied genes in 54 genes. Among them it is necessary to note genes encoding miRNA and «zinc fingers» involved in the regulation of transcription of many genes, transmembrane drug transporters and ion channels, genes of the MAP kinase signaling pathway, and others.Conclusion. The results of this study allow for the more exact determination of the expression picture of monoresistance genes in the tumor and the indication of new candidate genes which are involved in the regulation of the expression of these genes. Evaluation of the loss of heterozygosity in tumor tissue can be used as an additional criterion for personalizing chemotherapy. Цель исследования. Широкогеномное исследование связи потери гетерозиготности (loss of heterozygote, LOH) в опухоли молочной железы с экспрессией генов монорезистентности при проведении неоадъювантной химиотерапии (НХТ).Материал и методы. В исследование включены 68 больных раком молочной железы РМЖ IIA–IIIB стадий. РНК выделяли из опухолевого материала до и после лечения с помощью набора RNeasy mini kit Plus. Уровень экспрессии исследуемых генов (RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, BRCA1) оценивали при помощи RT-qPCR. ДНК выделяли из 68 биопсийных образцов опухолевой ткани с помощью набора QIAamp DNA mini Kit (Qiagen, Германия). Для оценки статуса LOH проведен микроматричный анализ на ДНК-чипах высокой плотности фирмы Affymetrix CytoScanTM HD Array.Результаты. В исследовании потеря гетерозиготности оценена у 13 815 генов. Частота LOH составила 0–63%. Наибольшая частота встречаемости явления потери гетерозиготности характерна для генов хромосом 16, 17 и Х-хромосомы. Установлено, что наличие явления LOH в генах монорезистентности (BRCA1, ERCC1, RRM1, TOP1, TOP2A, TUBB3 и TYMS) не сопряжено с их уровнем экспрессии в опухоли. Отмечено 54 гена, LOH в которых статистически значимо ассоциирована с уровнем экспрессии исследуемых генов. Среди них следует отметить гены, кодирующие miRNA и «цинковые пальцы», участвующие в регуляции транскрипции многих генов, трансмембранные транспортеры лекарственных препаратов и ионные каналы, гены MAP-киназного сигнального пути и др.Заключение. Результаты, полученные в данном исследовании, могут более точно определить экспрессионный портрет в опухоли генов монорезистентности, указать на новые кандидатные гены, участвующие в регуляции экспрессии этих генов. Оценка потери гетерозиготности в опухолевой ткани может быть использована в качестве дополнительного критерия для персонализации назначения химиотерапии.Purpose of work. To perform a genome-wide association study of loss of heterozygosity (LOH) with monoresistance genes expression during neoadjuvant chemotherapy (NAC) in breast cancer. Materials and methods. The study involved 68 patients with breast cancer. The tumour stages were IIAIIIB. RNA was extracted from tissue specimens (before and after NAC) using RNeasy Plus mini Kit (Qiagen, Germany). Expression profiling of the RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, BRCA1 genes was carried out using quantitative real-time PCR (qPCR). DNA was extracted from 68 biopsy specimens of tumour tissues using QIAamp DNA mini Kit (Qiagen, Germany). LOH status was detected using microarray analysis using high density DNA-chip manufactured by Affymetrix CytoScanTM HD Array company. Results. As a result of the study of loss of heterozygosity was evaluated in 13815 genes. The frequency of LOH varied from 0% to 63%. The highest incidence of heterozygosity loss events is characteristic for genes of 16, 17 and the X-chromosome. Our study established that the phenomenon of loss of heterozygosity in monoresistance genes (BRCA1, ERCC1, RRM1, TOP1, TOP2A, TUBB3 and TYMS), is not associated with their level of expression in the tumor. A statistical association has been found between LOH and level of expression of the studied genes in 54 genes. Among them it is necessary to note genes encoding miRNA and «zinc fingers» involved in the regulation of transcription of many genes, transmembrane drug transporters and ion channels, genes of the MAP kinase signaling pathway, and others. Conclusion. The results of this study allow for the more exact determination of the expression picture of monoresistance genes in the tumor and the indication of new candidate genes which are involved in the regulation of the expression of these genes. Evaluation of the loss of heterozygosity in tumor tissue can be used as an additional criterion for personalizing chemotherapy.