P78

Abstract

Breast cancer, particularly invasive carcinoma of no special type (IC NST), demonstrates considerable intratumor morphological heterogeneity. Five types of morphological structures representing different architectural arrangements of tumor cells – tubular, alveolar, trabecular, solid structures, and discrete groups have been described in IC NST. Previous studies reported the contribution of intratumor morphological heterogeneity of IC NST to chemotherapy efficiency and lymph node metastasis (Zavyalova et al., 2013; Denisov et al., 2014); however, its role in distant metastasis remains unidentified. Aim: to study the contribution of intratumor morphological heterogeneity of IC NST to distant metastasis and to identify gene expression features of metastatic behavior of different morphological structures. Materials and methods: 358 IC NST patients (age range 29–90, mean age 49.8 ± 9.5, T1-4N0-3M0-1) treated with neoadjuvant chemotherapy (NAC) have been enrolled in this study. Chi-square test and Kaplan–Meier analysis were used to estimate the association between the presence of certain morphological structures in breast tumors and the frequency of distant metastasis and metastasis-free survival. qRT-PCR was applied for measurement of the expression levels of genes involved in cell motility (CDH1, CDH2, CDH3, CTNNA1, CTNNB1, ITGA6, ITGAV, ITGB1, ITGB3, ITGB4, SNAIL, MMP14, ROCK2, L1CAM, MMP2, MMP9, PDPN) and pre-metastatic niche formation (TNFa, TGFb, VEGFa, LOX, M-CSF, GM-CSF, HIF1A, SDF2) in different morphological structures isolated from breast tumors (n = 4) by laser microdissection. Results: Patients with alveolar structures in breast tumors more frequently displayed distant metastasis than cases without this morphological variant (71.9% vs. 56.5%; p = 0.004). The association between alveolar structures and high frequency of hematogenous metastasis was found only in patients with poor response to NAC (p = 0.003), but not in cases with good chemotherapy efficiency (p = 0.377). Increased distant metastasis was also shown in patients with trabecular structures as compared to cases without this morphological type (88.3% vs. 70.0%; p = 0.0001). Kaplan–Meier analysis demonstrated a significantly higher probability of developing metastasis in patients with alveolar or trabecular structures in breast tumors (p = 0.011). No significant association between other morphological structures and distant metastasis was found. Expression analysis showed the presence of cell motility phenotype in all morphological structures. In particular, we found changes in cell adhesion gene expression, which declined in the row: solid–alveolar–trabecular structures–discrete groups of tumor cells (p < 0.05). In addition, almost all structures demonstrated SNAIL and ROCK2 gene expression, and there were differences in expression of other cell migration genes between morphological structures. For example, PDPN was observed to be expressed in solid and alveolar structures, whereas L1CAM – in trabecular, tubular structures and discrete groups of some breast tumors. The expression of pre-metastatic niche genes also varied between distinct structures and, in general, declined in the row: alveolar–solid–trabecular structures–discrete groups of tumor cells (p < 0.05). Conclusion: Intratumor morphological heterogeneity of IC NST contributes to distant metastasis probably by variations in expression of genes involved in cell motility and pre-metastatic niche formation between different morphological structures. The study was supported by the Russian Scientific Foundation (Grant #14-15-00318).