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. 2013 May 29;8(5):e64814.
doi: 10.1371/journal.pone.0064814. Print 2013.

High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor

Raoul A Droeser  1 , Christian Hirt, Serenella Eppenberger-Castori, Inti Zlobec, Carsten T Viehl, Daniel M Frey, Christian A Nebiker, Raffaele Rosso, Markus Zuber, Francesca Amicarella, Giandomenica Iezzi, Giuseppe Sconocchia, Michael Heberer, Alessandro Lugli, Luigi Tornillo, Daniel Oertli, Luigi Terracciano, Giulio C Spagnoli
Affiliations

High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor

Raoul A Droeser et al. PLoS One. .

Abstract

Background: Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG).

Methods: A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets.

Results: MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO-.

Conclusions: High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MPO and CD15 specific staining in CRC.
CRC samples were stained with MPO and CD15 specific monoclonal antibodies (clone 59A5, Novocastra and clone Carb-1, Leica, respectively). Tumor punches are representative of low (panels A and C) and high (panels B and D) density of CRC infiltrating MPO+ (panels A–B) and CD15+ (panels C–D) cells, respectively. Magnification: 20x. Panel E reports the distribution of MPO+ cells in normal mucosa (E1), total CRC (E2), MMR-deficient CRC (E3) and MMR-proficient CRC (E4). The green line indicates the cut-off of 60 cells/punch as defined by regression tree analysis. Panel F reports the distribution of CD15+ cells in normal mucosa (F1), total CRC (F2), MMR-deficient CRC (F3) and MMR-proficient CRC (F4). The green line indicates the cut-off of 46 cells/punch as defined by regression tree analysis.
Figure 2
Figure 2. Phenotypic characterization of CRC infiltrating MPO+ cells.
CRC surgical specimens were enzymatically digested and immediately stained with fluorochrome labeled mAbs recognizing MPO, HLA-DR, CD66b, CD15 and CD16, as indicated in “materials and methods”. Panel A reports one representative staining, whereas panel B summarizes results from freshly excised specimens (n = 8) regarding the expression of the indicated markers in CRC infiltrating MPO+ cells.
Figure 3
Figure 3. Effects of MPO+ and CD15+ tumor infiltration on overall survival in patients with CRC.
Kaplan-Meier overall survival curves were designed according to MPO+ and CD15+ tumor infiltration in patients bearing CRC. In panels A–C, dotted lines refer to high density and black lines to low density infiltration according to cut-off values established by regression tree analysis (60 cells/punch for MPO+ and 46 cells/punch for CD15+ cell infiltration). Panels A and B report the effects of high MPO+ cell infiltration, as detected in the training (n = 609; 255 deaths observed in 519 patients with low CRC infiltration by MPO+ cells and 28 deaths observed in 90 patients with tumors with high MPO+ cell infiltration, P = 0.038) and in the validation set (n = 583; 234 deaths observed in 498 patients with low CRC infiltration by MPO+ cells and 23 deaths observed in 85 patients with tumors with high MPO+ cell infiltration, P = 0.002). Panel C reports the effect of high CD15+ CRC infiltration in the whole group of patients under investigation (n = 1169; 458 deaths observed in 1041 patients with low CRC infiltration by CD15+ cells and 47 deaths observed in 128 patients with tumors with high CD15+ cell infiltration, P = 0.051). In panel D cumulative effects of tumor infiltration by MPO+ and CD15+ cells were explored. Rosa line (430/946) refers to tumors with low MPO+/CD15+ cell infiltration. Light blue line (18/34) refers to tumors with low MPO+ and high CD15+ cell infiltration. Lila line (29/91) refers to tumors with high MPO+/CD15+ cell infiltration and green line (18/76) refers to CRC with high MPO+ and low CD15+ cell infiltration (n = 1147; 430 deaths observed in 946 patients with low CRC infiltration by MPO+ and CD15+ cells; 18 deaths observed in 34 patients with tumors with high CD15+ and low MPO+ cell infiltration; 29 deaths observed in 91 patients with high CRC infiltration by MPO+ and CD15+ cells; 18 deaths observed in 76 patients with tumors with high MPO+ and low CD15+ cell infiltration, P = 0.002).
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Grants and funding

Financial support for this study was provided by the Swiss National Science Foundation (SNF) Grants Nr. PP00P3-133699, Nr. 31003A-122235 and Nr. 320030-120320, the Italian Association for Cancer Research (AIRC) IG Grant Nr. 10555, the Rainbow Association for Research in Pediatric Oncology-Hematology/The NANDO PERETTI Foundation, and Lazio Regional Agency for Transplantation and Related Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.