Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

<div><p>We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, <em>in vitro</em> analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.</p> </div

Demographic and clinical characteristics of study participants.

<p>N: number of individuals; N.A.: not applicable; N.D.: not determined; UICC: Union for International Cancer Control staging.</p>*<p>Patient with localized CRC was diagnosed based on biopsy material and computed tomography scan. Stage of disease was not determined, as patient did not undergo surgery.</p

CD16 expression is increased on monocytes of cancer patients.

<p>The expression level of CD16 and TIE2 on monocyte subsets was determined for healthy individuals and patients with localized or metastatic colorectal cancer. Mean fluorescence intensity (MFI) of CD16-PC5 staining was evaluated for intermediate CD14++CD16+ monocytes (A), and CD14++CD16+TIE2+ TEMs (B). TIE2 expression levels are reflected by MFI of TIE2-PE antibody bound to TEMs (C).</p

Blood levels of intermediate monocytes are a diagnostic indicator of CRC.

<p>The balance in diagnostic sensitivity and specificity of intermediate monocyte levels was evaluated by ROC chart and the area under the curve.</p

Univariate and multivariate analysis by binary logistic regression to calculate significance levels and odds ratios (including 95% confidence intervals) for cancer prediction by the investigated variables.

<p>Univariate and multivariate analysis by binary logistic regression to calculate significance levels and odds ratios (including 95% confidence intervals) for cancer prediction by the investigated variables.</p

CD14 expression of all monocyte populations decreases in the advanced stage of mCRC.

<p>CD14 expression levels in healthy individuals, in patients with localized CRC or mCRC were assessed by mean fluorescence intensity (MFI) of CD14-FITC staining on total (CD14 positive) monocytes (A) or on the subsets of classical CD14++CD16− monocytes (B), intermediate CD14++CD16+ monocytes (C), and CD14++CD16+TIE2+ TEMs (D).</p

Intermediate monocytes are significantly elevated in CRC patients.

<p>Monocyte subpopulations were determined by flow cytometry and are given in % of total leukocyte counts in healthy individuals, patients with localized CRC and patients with mCRC. The frequency of total (CD14 positive) monocytes (A) was compared to classical CD14++CD16- monocytes (B), intermediate CD14++CD16+ monocytes (C), and CD14++CD16+TIE2+ TEMs (D).</p