Abstract B21: Macrophages and T cell infiltration in colorectal liver metastases according to growth pattern
Authors: Eefsen Rikke L. , Eynden Gert G. , Alpizar-Alpizar Warner , Høyer-Hansen Gunilla , Christensen Ib Jarle , Engelholm Lars , Lærum Ole Didrik , Vermeulen Peter B. , Østerlind Kell , Vainer Ben , Illemann Martin 
Published: August 2014

Metastatic disease is one of the major challenges in the treatment of colorectal cancer (CRC). Three different growth patterns of CRC liver metastases (CLM) have been identified, i.e. desmoplastic, pushing and replacement. The desmoplastic growth pattern represent the best prognostic outcome. Previously we demonstrated that growth patterns differ in their biological properties, such as angiogenesis and plasminogen activation. Furthermore, we demonstrated that multiple colorectal liver metastases resected from one patient often represented the same growth pattern. These findings indicate, that invasion and growth of CLMs are governed by different mechanisms. The aim of this study was to deduce if the macrophage and T-cell infiltration differed among the three growth patterns.

This study included twenty-two chemonaive patients resected for multiple CLMs, represented by 56 metastases. Macrophages were identified by a monoclonal antibody against CD68 and a monoclonal CD3 antibody was used to identify T-cells. Reticulin and HE staining were used for the evaluation the CLM growth pattern. Quantification of CD68 was performed by light microscopy (400x) of five randomly selected fields at the invasive front, as well as by digital image analysis, using Visiopharm software. All slides were scanned by a Mirax Midi Scanner. Region of Interest (ROI) was identified on each slide by inclusion of 10-15 cell rows at each side of the invasive front. In twenty of the slides ROIs were drawn by two scientists. The digital evaluation was done with a digital classifier for CD68. Evaluation of T-cell infiltration was done by the digital image analysis.

Manual score of CD68 at the invasive front showed an upregulation of 52% in desmoplastic CLMs in comparison to pushing (95% CI: 19-93%, p=0.001). Digital image analysis of CD68 supported the manual score by 41% upregulation in desmoplastic CLMs in comparison to pushing (95% CI: 12-78%, p=0.004). Macrophages were also upregulated in replacement CLMs in comparison to pushing, both by manual score (34%, 95% CI: 26-77%, p=0.032) and digital score (74%, 95% CI: 34-125%, p=&lt;.0001). The measurement between two observers by digital analysis, had a systematic difference of 10%, however inter observer correlation was high (rs=0.90). Correlation between manual and digital score was r=0.63. The number of T-cells was significantly higher in replacement CLMs than in CLMs of pushing (42%, 95% CI: 13-62%, p=0.01), but not when compared to desmoplastic CLMs (17%, 95% CI: 46-73%, p=0.38). T-cell infiltration was more pronounced at the invasive front of the desmoplastic CLMs in comparison to pushing (43%, 95% CI: 1-150%, p=0.054), but this finding was not significant.

Infiltration of macrophages and T-cells at the invasive front differ among the three CLM growth patterns. Macrophages and T-cells seem to be involved in the invasion of liver metastases with desmoplastic and replacement growth, whereas only a small amount of immune cells are present in CLMs with a pushing growth pattern. Invasion of pushing CLMs may be directly facilitated by the cancer cells or in association with cells other than macrophages and T-cells. The different biological properties of CLMs may be of major clinical relevance.

Citation Format: Rikke L. Eefsen, Gert G. Eynden, Warner Alpizar-Alpizar, Gunilla Høyer-Hansen, Ib Jarle Christensen, Lars Engelholm, Ole Didrik Lærum, Peter B. Vermeulen, Kell Østerlind, Ben Vainer, Martin Illemann. Macrophages and T cell infiltration in colorectal liver metastases according to growth pattern. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B21.