Subtype-Based Prognostic Analysis of Cell-in-Cell Structures in Early Breast Cancer

Xin Zhang, Zubiao Niu, Hongquan Qin, Jie Fan, Manna Wang, Bo Zhang, You Zheng, Lihua Gao, Zhaolie Chen, Yanhong Tai, Mo Yang, Hongyan Huang, Qiang Sun, Xin Zhang, Zubiao Niu, Hongquan Qin, Jie Fan, Manna Wang, Bo Zhang, You Zheng, Lihua Gao, Zhaolie Chen, Yanhong Tai, Mo Yang, Hongyan Huang, Qiang Sun

Abstract

Though current pathological methods are greatly improved, they provide rather limited functional information. Cell-in-cell structures (CICs), arising from active cell-cell interaction, are functional surrogates of complicated cell behaviors within heterogeneous cancers. In light of this, we performed the subtype-based CIC profiling in human breast cancers by the "EML" multiplex staining method, and accessed their values as prognostic factors by Cox univariate, multivariate, and nomogram analysis. CICs were detected in cancer specimens but not in normal breast tissues. A total of five types of CICs were identified with one homotypic subtype (91%) and four heterotypic subtypes (9%). Overall CICs (oCICs) significantly associated with patient overall survival (OS) (P = 0.011) as an independent protective factor (HR = 0.423, 95% CI, 0.227-0.785; P = 0.006). Remarkably, three CICs subtypes (TiT, TiM, and MiT) were also independent prognostic factors. Among them, higher TiT, from homotypic cannibalism between tumor cells, predicted longer patient survival (HR = 0.529, 95% CI, 0.288-0.973; P = 0.04) in a way similar to that of oCICs and that (HR = 0.524, 95% CI, 0.286-0.962; P = 0.037) of heterotypic TiM (tumor cell inside macrophage); conversely, the presence of MiT (macrophage inside tumor cell) predicted a death hazard of 2.608 (95% CI, 1.344-5.063; P = 0.05). Moreover, each CIC subtype tended to preferentially affect different categories of breast cancer, with TiT (P < 0.0001) and oCICs (P = 0.008) targeting luminal B (Her2+), TiM (P = 0.011) targeting HR- (Her2+/HR- and TNBC), and MiT targeting luminal A (P = 0.017) and luminal B (Her-) (P = 0.006). Furthermore, nomogram analysis suggested that CICs impacted patient outcomes in contributions comparable (for oCICs, TiT, and TiM), or even superior (for MiT), to TNM stage and breast cancer subtype, and incorporating CICs improved nomogram performance. Together, we propose CICs profiling as a valuable way for prognostic analysis of breast cancer and that CICs and their subtypes, such as MiT, may serve as a type of novel functional markers assisting clinical practices.

Keywords: breast cancer; cell-in-cell structures; macrophage; overall survival; prognosis.

Copyright © 2019 Zhang, Niu, Qin, Fan, Wang, Zhang, Zheng, Gao, Chen, Tai, Yang, Huang and Sun.

Figures

Figure 1
Figure 1
Cell-in-cell structures in breast cancer. (A) Representative image for CICs in human breast cancer tissue stained with antibodies for E-cadherin. Nuclei were counterstained with DAPI. Right panel shows zoomed images for boxed region in the left image. Yellow arrows indicate inner cells of cell-in-cell structures. Scale bar: 20 μm. (B) Distribution of overall CICs (oCICs) across breast cancer tissues from different patients.
Figure 2
Figure 2
Subtype profiling of cell-in-cell structures in breast cancer. (A–E) Representative images for five CIC subtypes as indicated. Right panel of pictures demonstrate the schematic structure for each subtype of CICs. Scale bar: 10 or 20 μm; (F) composition analysis of five CIC subtypes; (G) distribution of five CIC subtypes across breast cancer tissues from different patients.
Figure 3
Figure 3
Cell-in-cell structures associate with overall survival (OS) of breast cancer patients. Kaplan–Meier plotting for OS curves of oCICs (A), TiT (B), TiM (C), and MiT (D). High for CICs ≥ 15/core, low for CICs < 15/core; (–) and (+) for cores negative or positive for CICs, respectively.
Figure 4
Figure 4
Survival impacts of subtyped cell-in-cell structures on different categories of breast cancers. Kaplan–Meier analyses of prognostic values of oCICs (A–F), TiT (G–L), TiM (M–R), and MiT (S–X) in five categories/subtypes of breast cancer. High for CICs ≥ 15/core, low for CICs < 15/core; (–) and (+) for cores negative or positive for CICs, respectively.
Figure 5
Figure 5
Contribution of subtyped cell-in-cell structures to survival prediction by nomogram analysis. (A) Prognostic nomogram in the absence of CICs as a viable. (B–E) Prognostic nomogram with oCICs (B), or TiT (C), or TiM (D), or MiT (E) as a variable, respectively. For the probability of patient survival for 84.25 months, summing up the points from individual variable, locating its position on the axis of total points to determine the corresponding survival probability based on the bottom line (survival at 84.25) of the nomogram. In addition to CICs, age, TNM stage, ER and PR statuses, and cancer subtypes were included in the nomogram analysis. (F) The AUC of different nomogram models from observed data.

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