Anne Tuomisto

Stage-dependent alterations of the serum cytokine pattern in colorectal carcinoma

Background:Inflammation contributes to the pathogenesis of colorectal cancer (CRC), and cytokine levels are altered during colorectal carcinogenesis.Methods:The serum levels of 13 cytokines and their relation to clinical and pathological parameters, and systemic inflammatory response (mGPS, CRP and neutrophil–lymphocyte ratio), were analysed from a prospective series of 148 CRC patients and 86 healthy age- and sex-matched controls.Results:CRC patients had higher serum platelet-derived growth factor, interleukin (IL)-6, IL-7, and IL-8 levels and lower monocyte chemotactic protein-1 (MCP-1) levels than the controls. A logistic regression model for discriminating the patients from the controls – including the five most predictive cytokines (high IL-8, high IL-6, low MCP-1, low IL-1ra, and low IP-10) – yielded an area under curve value of 0.890 in receiver operating characteristics analysis. Serum cytokines showed distinct correlation with other markers of systemic inflammatory response, and advanced CRCs were associated with higher levels of IL-8, IL-1ra, and IL-6. A metastasised disease was accompanied by an orientation towards Th2 cytokine milieu.Conclusion:CRC is associated with extensive alterations in serum cytokine environment, highlighting the importance of studying relative cytokine level alterations. Serum cytokine profile shows promise in separating CRC patients from healthy controls but its clinical value is yet to be confirmed.

Type XIII collagen: a novel cell adhesion component present in a range of cell–matrix adhesions and in the intercalated discs between cardiac muscle cells

Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell-basement membrane interfaces. Some cell-cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell-matrix junctions.

Detailed analysis of inflammatory cell infiltration in colorectal cancer

Background:Higher-grade inflammatory infiltrate is a promising marker for better prognosis in colorectal cancer (CRC). However, the knowledge on the interrelationships between different inflammatory cells and classifications is fragmentary.Methods:We analysed the densities of eight types of inflammatory cells in a prospectively recruited group of 117 CRC patients and determined their interrelationships and contributions to Klintrup–Mäkinen (K–M) score of overall peritumoural inflammation. We characterised the inflammatory infiltrate in relation to stage and recurrences in 24-month follow-up.Results:There were high positive correlations between the inflammatory cell densities, with the exception of mast cells and CD1a+ immature dendritic cells. High K–M score associated with high peri- and intratumoural densities of CD3+, CD8+, CD68+, CD83+, and FoxP3+ cells and neutrophils. Advanced stage associated with low K–M score, as well as low CD3+, CD8+, CD83+, and FoxP3+ cell counts, of which low K–M score, low CD3+ T-cell count, and low FoxP3+ T-cell count were linked to higher recurrence rate.Conclusion:The density of CRC inflammatory infiltrate declines as stage advances. Especially, low K–M score and low T-cell counts predict higher recurrence rate. The high positive correlations between the individual inflammatory markers support the value of overall inflammatory reaction scoring.

Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma

Stefanius K, Ylitalo L, Tuomisto A, Kuivila R, Kantola T, Sirniö P, Karttunen T J & Mäkinen M J
(2011) Histopathology58, 679–692
Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma

Characteristics and significance of colorectal cancer associated lymphoid reaction.

A subset of colorectal cancers (CRCs) exhibits so‐called Crohns like lymphoid reaction (CLR), an inflammatory reaction pattern that consists of numerous transmural lymphoid aggregates. However, the composition of these aggregates, their biological mechanisms and their prognostic significance are not well‐defined. We analyzed two CRC cohorts (418 and 149 patients) and determined clinicopathological features including survival. A new method for evaluating CLR based on counting the areal density of the lymphoid follicles (CLR density) was adopted. Immune cell densities at intratumoral and peritumoral regions, as well as the composition of the lymphoid follicles, were studied by immunohistochemistry. We found that CLR comprised of lymphoid aggregates with no evidence of granuloma formation. High CLR density associated with lower tumor stage, lack of preoperative radiotherapy or chemoradiotherapy and deficient mismatch repair enzyme expression. CLR density had positive correlations with peritumoral and intratumoral densities of CD83+ mature dendritic cells and T cells. High CLR density associated with better survival and had prognostic value that was independent of stage, Klintrup‐Mäkinen score for peritumoral inflammation and the numbers of tumor infiltrating T cells. CLR density evaluation had excellent intraobserver and interobserver agreement. In conclusion, the results suggest that CLR contributes to the adaptive antitumor immunity. Quantitative evaluation of CLR density is a relevant prognostic indicator in CRC.

Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated α1(XIII) chains. Shortened α1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.

Serum MMP-8 levels increase in colorectal cancer and correlate with disease course and inflammatory properties of primary tumors.

Matrix metalloproteinases (MMPs) form a family of zinc‐dependent endoproteases participating in cancer pathogenesis by promoting invasion and regulating growth signaling, apoptosis, angiogenesis and immune responses. MMP‐8 is an intriguing MMP with recently discovered antitumor activity and immunoregulatory properties, but its role in colorectal cancer (CRC) has not been studied extensively. Preoperative serum MMP‐8 levels (S‐MMP‐8) of 148 CRC patients and 83 healthy controls were measured using an immunofluorometric assay and related to clinical and pathological parameters. The patients had higher S‐MMP‐8 than the controls (median 63.0 vs. 17.2 ng/ml, p = 1.5E − 9), and a receiver operating characteristics analysis yielded an area under the curve of 0.751 in differentiating the groups. In univariate analyses, S‐MMP‐8 correlated positively with disease stage (p = 4.5E − 4), the degree of primary tumor necrosis (p = 0.0024) and blood neutrophil count (Pearson r = 0.523, p = 2.5E − 9). Particular interest was also addressed to the inflammatory properties of the tumors, and both variables studied, peritumoral tumor‐destructing inflammatory infiltrate and Crohns‐like lymphoid reaction (CLR), showed a negative correlation with S‐MMP‐8 (p = 0.041 and p = 0.0057, respectively). In a multiple linear regression analysis, high S‐MMP‐8 associated with elevated blood neutrophil count, distant metastases, low‐grade CLR and low body mass index. Overall, our results indicate that MMP‐8 is involved in the course and progression of CRC influencing the immune response against the tumor and contributing to the resolution of necrosis. Serum or plasma MMP‐8 may prove to be a worthy biomarker for CRC.

An improved image analysis method for cell counting lends credibility to the prognostic significance of T cells in colorectal cancer

Numerous immunohistochemically detectable proteins, such as immune cell surface (CD) proteins, vascular endothelial growth factor, and matrix metalloproteinases, have been proposed as potential prognostic markers in colorectal cancer (CRC) and other malignancies. However, the lack of reproducibility has been a major problem in validating the clinical use of such markers, and this has been attributed to insufficiently robust methods used in immunohistochemical staining or its assessment. In this study, we assessed how computer-assisted image analysis might contribute to the reliable assessment of positive area percentage and immune cell density in CRC specimens, and subsequently, we applied the computer-assisted cell counting method in assessing the prognostic value of T cell infiltration in CRC. The computer-assisted analysis methods were based on separating hematoxylin and diaminobenzidine color layers and then applying a brightness threshold using open source image analysis software ImageJ. We found that computer-based analysis results in a more reproducible assessment of the immune positive area percentage than visual semiquantitative estimation. Computer-assisted immune cell counting was rapid to perform and accurate (Pearson r > 0.96 with exact manual cell counts). Moreover, the computer-assisted determination of peritumoral and stromal T cell density had independent prognostic value. Our results suggest that computer-assisted image analysis, utilizing freely available image analysis software, provides a valuable alternative to semiquantitative assessment of immunohistochemical results in cancer research, as well as in clinical practice. The advantages of using computer-assisted analysis include objectivity, accuracy, reproducibility, and time efficiency. This study supports the prognostic value of assessing T cell infiltration in CRC.

Serum endostatin levels are elevated in colorectal cancer and correlate with invasion and systemic inflammatory markers.

Background:Endostatin, a fragment of collagen XVIII, is an endogenous angiogenesis inhibitor with anti-tumour functions. However, elevated circulating endostatin concentrations have been found in several human cancers including colorectal cancer (CRC).Methods:Serum endostatin levels were measured by enzyme-linked immunoassay from a series of 143 patients with CRC and from 84 controls, and correlated with detailed clinicopathological features of CRC, serum leukocyte differential count and C-reactive protein (CRP) levels.Results:Patients with CRC had higher serum endostatin levels than the controls (P=0.005), and high levels associated with age, tumour invasion through the muscularis propria and poor differentiation, but not with metastases. Endostatin levels showed a positive correlation with the markers of systemic inflammatory response and a negative correlation with the densities of tumour-infiltrating mast cells and dendritic cells. Collagen XVIII was expressed in tumour stroma most strikingly in blood vessels and capillaries, and in the muscle layer of the bowel wall.Conclusions:Elevated endostatin levels in CRC correlate with systemic inflammation and invasion through the muscularis propria. Increased endostatin level may be a result of invasion-related cleavage of collagen XVIII expressed in the bowel wall. The negative correlations between serum endostatin and intratumoural mast cells and immature dendritic cells may reflect angiogenesis inhibition by endostatin.

The relationships between serum cytokine levels and tumor infiltrating immune cells and their clinical significance in colorectal cancer

Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease‐free survival (DFS), cancer‐specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL‐12 levels associated with increased densities of peritumoral CD8+ T cells, intraepithelial CD3+ T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68+ cells. In multivariate survival models, increased infiltration of intraepithelial CD3+ T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83+ dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3+ T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3+ T cells, CD83+ dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome.