Maria L. Wikberg

The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer

High macrophage infiltration has been correlated to improved survival in colorectal cancer (CRC). Tumor associated macrophages (TAMs) play complex roles in tumorigenesis since they are believed to hold both tumor preventing (M1 macrophages) and tumor promoting (M2 macrophages) activities. Here we have applied an immunohistochemical approach to determine the degree of infiltrating macrophages with a M1 or M2 phenotype in clinical specimens of CRC in relation to prognosis, both in CRC in general but also in subgroups of CRC defined by microsatellite instability (MSI) screening status and the CpG island methylator phenotype (CIMP). A total of 485 consecutive CRC specimens were stained for nitric oxide synthase 2 (NOS2) (also denoted iNOS) as a marker for the M1 macrophage phenotype and the scavenger receptor CD163 as a marker for the M2 macrophage phenotype. The average infiltration of NOS2 and CD163 expressing macrophages along the invasive tumor front was semi-quantitatively evaluated using a four-graded scale. Two subtypes of macrophages, displaying M1 (NOS2+) or M2 (CD163+) phenotypes, were recognized. We observed a significant correlation between the amount of NOS2+ and CD163+ cells (P<0.0001). A strong inverse correlation to tumor stage was found for both NOS2 (P<0.0001) and CD163 (P<0.0001) infiltration. Furthermore, patients harbouring tumors highly infiltrated by NOS2+ cells had a significantly better prognosis than those infiltrated by few NOS2+ cells, and this was found to be independent of MSI screening status and CIMP status. No significant difference was found on cancer-specific survival in groups of CRC with different NOS2/CD163 ratios. In conclusion, an increased infiltration of macrophages with a M1 phenotype at the tumor front is accompanied by a concomitant increase in macrophages with a M2 phenotype, and in a stage dependent manner correlated to a better prognosis in patients with CRC.

The prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer

BackgroundMutations in KRAS, BRAF, PIK3CA and PTEN expression have been in focus to predict the effect of epidermal growth factor receptor-blocking therapy in colorectal cancer (CRC). Here, information on these four aberrations was collected and combined to a Quadruple index and used to evaluate the prognostic role of these factors in CRC.PatientsWe analysed the mutation status in KRAS, BRAF and PIK3CA and PTEN expression in two separate CRC cohorts, Northern Sweden Health Disease Study (NSHDS; n=197) and Colorectal Cancer in Umeå Study (CRUMS; n=414). A Quadruple index was created, where Quadruple index positivity specifies cases with any aberration in KRAS, BRAF, PIK3CA or PTEN expression.ResultsQuadruple index positive tumours had a worse prognosis, significant in the NSHDS but not in the CRUMS cohort (NSHDS; P=0.003 and CRUMS; P=0.230) in univariate analyses but significance was lost in multivariate analyses. When analysing each gene separately, only BRAF was of prognostic significance in the NSHDS cohort (multivariate HR 2.00, 95% CI: 1.16–3.43) and KRAS was of prognostic significance in the CRUMS cohort (multivariate HR 1.48, 95% CI: 1.02–2.16). Aberrations in PIK3CA and PTEN did not add significant prognostic information.ConclusionsOur results suggest that establishment of molecular subgroups based on KRAS and BRAF mutation status is important and should be considered in future prognostic studies in CRC.

The intratumoural subsite and relation of CD8(+) and FOXP3(+) T lymphocytes in colorectal cancer provide important prognostic clues

Background:To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8+) and regulatory T lymphocytes (FoxP3+) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status).Methods:CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8+ and FOXP3+ cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial).Results:We found that infiltration of CD8+ T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3+ T-lymphocyte infiltration to be a better prognostic tool than CD8+ T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3+ cells at the tumour invasive front, significantly improved prognosis.Conclusions:Analyses of intraepithelial infiltration of CD8+ T lymphocytes, infiltration of FOXP3+ T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.

Phenotypic skewing of macrophages in vitro by secreted factors from colorectal cancer cells.

Macrophages are cells with many important functions in both innate and adaptive immune responses and have been shown to play a complex role in tumor progression since they harbour both tumor preventing (M1 macrophages) and tumor promoting (M2 macrophages) activities. In many human cancers, infiltrating macrophages have been associated with a poor patient prognosis, and therefore suggested to be mainly of an M2 phenotype. However, we and others have previously shown that increased macrophage density in colorectal cancer (CRC) instead is correlated with an improved prognosis. It is an intriguing question if the different roles played by macrophages in various cancers could be explained by variations in the balance between M1 and M2 macrophage attributes, driven by tumor- or organ-specific factors in the tumor microenvironment of individual cancers. Here, we utilized an in vitro cell culture system of macrophage differentiation to compare differences and similarities in the phenotype (morphology, antigen-presentation, migration, endocytosis, and expression of cytokine and chemokine genes) between M1/M2 and tumor activated macrophages (TAMs), that could explain the positive role of macrophages in CRC. We found that secreted factors from CRC cells induced TAMs of a “mixed” M1/M2 phenotype, which in turn could contribute to a “good inflammatory response”. This suggests that re-education of macrophages might allow for important therapeutic advances in the treatment of human cancer.

SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer

BackgroundTo improve current treatment strategies for patients with aggressive colorectal cancer (CRC), the molecular understanding of subgroups of CRC with poor prognosis is of vast importance. SOX2 positive tumors have been associated with a poor patient outcome, but the functional role of SOX2 in CRC patient prognosis is still unclear.MethodsAn in vitro cell culture model expressing SOX2 was used to investigate the functional role of SOX2 in CRC. In vitro findings were verified using RNA from fresh frozen tumor tissue or immunohistochemistry on formalin fixed paraffin embedded (FFPE) tumor tissue from a cohort of 445 CRC patients.ResultsUsing our in vitro model, we found that SOX2 expressing cells displayed several characteristics of cancer stem cells; such as a decreased proliferative rate, a spheroid growth pattern, and increased expression of stem cell markers CD24 and CD44. Cells expressing SOX2 also showed down-regulated expression of the intestinal epithelial marker CDX2. We next evaluated CDX2 expression in our patient cohort. CDX2 down-regulation was more often found in right sided tumors of high grade and high stage. Furthermore, a decreased expression of CDX2 was closely linked to MSI, CIMP-high as well as BRAF mutated tumors. A decreased expression of CDX2 was also, in a stepwise manner, strongly correlated to a poor patient prognosis. When looking at SOX2 expression in relation to CDX2, we found that SOX2 expressing tumors more often displayed a down-regulated expression of CDX2. In addition, SOX2 expressing tumors with a down-regulated CDX2 expression had a worse patient prognosis compared to those with retained CDX2 expression.ConclusionsOur results indicate that SOX2 expression induces a cellular stem cell state in human CRC with a decreased expression of CDX2. Furthermore, a down-regulated expression of CDX2 results in a poor patient prognosis in CRC and at least part of the prognostic importance of SOX2 is mediated through CDX2 down-regulation.

Secreted Factors from Colorectal and Prostate Cancer Cells Skew the Immune Response in Opposite Directions.

Macrophage infiltration has been associated with an improved prognosis in patients with colorectal cancer (CRC), but a poor prognosis in prostate cancer (PC) patients. In this study, the distribution and prognostic value of proinflammatory M1 macrophages (NOS2+) and immunosuppressive M2 macrophages (CD163+) was evaluated in a cohort of 234 PC patients. We found that macrophages infiltrating PC were mainly of an M2 type and correlated with a more aggressive tumor and poor patient prognosis. Furthermore, the M1/M2 ratio was significantly decreased in PC compared to CRC. Using in vitro cell culture experiments, we could show that factors secreted from CRC and PC cells induced macrophages of a proinflammatory or immunosuppressive phenotype, respectively. These macrophages differentially affected autologous T lymphocyte proliferation and activation. Consistent with this, CRC specimens were found to have higher degrees of infiltrating T-helper 1 cells and active cytotoxic T lymphocytes, while PC specimens displayed functionally inactive T cells. In conclusion, our results imply that tumour-secreted factors from cancers of different origin can drive macrophage differentiation in opposite directions and thereby regulate the organization of the anti-tumour immune response. Our findings suggest that reprogramming of macrophages could be an important tool in the development of new immunotherapeutic strategies.

Macrophages: Good guys in colorectal cancer.

Macrophages play a complex role in tumor progression since they can exert both tumor-preventing (M1 macrophages) and tumor-promoting (M2 macrophages) activities. In colorectal carcinoma (CRC), at odds to many other cancers, macrophage infiltration has been correlated with an improved patient survival. In a recent study, we have evaluated the distribution of M1 and M2 macrophage subtypes in CRC and their impact on patient prognosis.

SOX2 Expression Is Regulated by BRAF and Contributes to Poor Patient Prognosis in Colorectal Cancer

Sporadic colorectal cancer (CRC) is a common malignancy and also one of the main causes of cancer deaths worldwide. Aberrant expression of the transcription factor SOX2 has recently been observed in several cancer types, but its role in CRC has not been fully elucidated. Here we studied the expression of SOX2 in 441 CRC patients by immunohistochemistry and related the expression to clinicopathological and molecular variables and patient prognosis. SOX2 was expressed in 11% of the tumors and was significantly associated to BRAFV600E mutation, but not to KRAS mutations (codon 12 and 13). SOX2 positivity was correlated to poor patient survival, especially in BRAFV600E mutated cases. In vitro studies showed that cells expressing the constitutively active BRAFV600E had increased SOX2 expression, a finding not found in cells expressing KRASG12V. Furthermore, blocking downstream BRAF signalling using a MEK-inhibitor resulted in a decreased expression of SOX2. Since SOX2 overexpression has been correlated to increased migration and invasion, we investigated the SOX2 expression in human CRC liver metastasis and found that a SOX2 positive primary CRC also had SOX2 expression in corresponding liver metastases. Finally we found that cells overexpressing SOX2 in vitro showed enhanced expression of FGFR1, which has been reported to correlate with liver metastasis in CRC. Our novel findings suggest that SOX2 expression is partly regulated by BRAF signalling, and an increased SOX2 expression may promote CRC metastasis and mediate a poor patient prognosis.

Cancer-associated fecal microbial markers in colorectal cancer detection

Colorectal cancer (CRC) is the second most common cause of cancer death in the western world. An effective screening program leading to early detection of disease would severely reduce the mortality of CRC. Alterations in the gut microbiota have been linked to CRC, but the potential of microbial markers for use in CRC screening has been largely unstudied. We used a nested case–control study of 238 study subjects to explore the use of microbial markers for clbA+ bacteria harboring the pks pathogenicity island, afa‐C+ diffusely adherent Escherichia coli harboring the afa‐1 operon, and Fusobacterium nucleatum in stool as potential screening markers for CRC. We found that individual markers for clbA+ bacteria and F. nucleatum were more abundant in stool of patients with CRC, and could predict cancer with a relatively high specificity (81.5% and 76.9%, respectively) and with a sensitivity of 56.4% and 69.2%, respectively. In a combined test of clbA+ bacteria and F. nucleatum, CRC was detected with a specificity of 63.1% and a sensitivity of 84.6%. Our findings support a potential value of microbial factors in stool as putative noninvasive biomarkers for CRC detection. We propose that microbial markers may represent an important future screening strategy for CRC, selecting patients with a “high‐risk” microbial pattern to other further diagnostic procedures such as colonoscopy.

The Association of Immune Cell Infiltration and Prognosis in Colorectal Cancer

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. The tumor microenvironment is of great biological importance in cancer development and progression and harbors several different immune cells representing both the innate and the adaptive immune response. These inflammatory cells may have both tumor-promoting and tumor-suppressing effects, and they have been shown to be of prognostic importance. This review summarizes the recent evidence regarding the prognostic significance of the host response to colorectal cancer.