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.

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

BACKGROUND: To 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. METHODS: An 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. RESULTS: Using 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. CONCLUSIONS: Our 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.

High intratumoral expression of fibroblast activation protein (FAP) in colon cancer is associated with poorer patient prognosis.

An active stroma is important for cancer cell invasion and metastasis. We investigated the expression of fibroblast activation protein (FAP) in relation to patient prognosis in colorectal cancer. Colorectal cancer specimens from 449 patients were immunohistochemically stained with a FAP antibody and evaluated in the tumor center and tumor front using a semiquantitative four-level scale. FAP was expressed by fibroblasts in 85-90 % of the tumors examined. High versus no/low expression in the tumor center was associated with poor prognosis (multivariate hazard ratio, HR = 1.72; 95 % CI 1.07-2.77, p = 0.025). FAP expression in the tumor front, though more frequent than in the tumor center, was not associated with prognosis. FAP expression in the tumor center was more common in specimens with positive microsatellite instability (MSI) screening status and in patients with high CpG island methylator phenotype (CIMP) status. However, inclusion of MSI screening status and CIMP status in the multivariate analysis strengthened the risk estimates for high FAP expression in the tumor center (HR = 1.89; 95 % CI 1.13-3.14; p = 0.014), emphasizing the role of FAP as an independent prognostic factor. Stromal FAP expression is common in colorectal cancer, and we conclude that high FAP expression in the tumor center, but not the tumor front, is an independent negative prognostic factor.

Plasma miRNA can detect colorectal cancer, but how early?

Colorectal cancer (CRC) is a major cause of deaths worldwide but has a good prognosis if detected early. The need for efficient, preferable non- or minimally invasive, inexpensive screening tools is therefore critical. We analyzed 12 miRNAs in pre- and postdiagnostic plasma samples to evaluate their potential as CRC screening markers. We used a unique study design with two overlapping cohorts, allowing analysis of pre- and postdiagnostic samples from 58 patients with CRC and matched healthy controls. Plasma concentrations of miR-15b, -16, -18a, -19a, 21, -22, -25, -26a, -29c, -142-5p, -150, and -192 were measured by semi-quantitative real-time PCR. Concentrations of miR-18a, -21, -22, and -25 in plasma from patients with CRC were significantly altered compared to healthy controls. Combined as a multimarker panel, they detected CRC with an AUC of 0.93. Furthermore, levels of these three miRNAs also showed different levels in the prediagnostic case samples close to diagnosis. Only miR-21-levels were elevated several years before diagnosis. Plasma levels of miR-18a, -21, -22, and -25 show promise as screening biomarkers for CRC. However, based on our unique analysis of prediagnostic and postdiagnostic samples from the same patients, we conclude that circulating miRNAs elevated at diagnosis may not automatically be suitable for CRC screening, if the increase occurs too close to clinical diagnosis.

MicroRNA Expression in KRAS- and BRAF-mutated Colorectal Cancers.

BACKGROUND/AIM: KRAS and BRAF are two genes commonly mutated in colorectal cancer (CRC). Even though BRAF is a downstream target of KRAS in the MAPK signalling pathway, KRAS- and BRAF-mutated CRCs are found to display several different clinical and histopathological features. We investigated whether a differential expression of microRNAs (miRNAs) could explain the clinicopathological differences seen between KRAS- and BRAF-mutated CRCs. MATERIALS AND METHODS: Using a PCR array, we analyzed the expression of 84 different miRNAs in CRC cell lines wild-type in KRAS and BRAF, or mutated in KRAS or BRAF. RESULTS: Ten miRNAs were selected for further analyses in tumor tissue specimens (let-7a, let-7i, miR-10a, miR-10b, miR-31, miR-100, miR-181a, miR-181b, miR-372, and miR-373). BRAF-mutated tumors were found to express significantly higher levels of miR-31 as well as significantly lower levels of miR-373, compared to wild-type tumors. CONCLUSION: Our results suggest that KRAS- and BRAF-mutated CRCs may have different miRNA signatures compared to CRC tumors wild-type in KRAS and BRAF. However, no difference in expression levels between KRAS- and BRAF-mutated tumors was evident for the miRNAs analyzed in this study.

Neutrophil infiltration is a favorable prognostic factor in early stages of colon cancer.

The tumor immune response has been proven critical to prognosis in colorectal cancer (CRC), but studies on the prognostic role of neutrophil infiltration have shown contradictory results. The aim of this study was to elucidate the prognostic role of infiltrating neutrophils at different intratumoral subsites and in different molecular subgroups of CRC. The relations between neutrophil infiltration and infiltration of other immune cells (T-cell and macrophage subsets) were also addressed. Expression of the neutrophil marker CD66b was assessed by immunohistochemistry in 448 archival human tumor tissue samples from patients surgically resected for CRC. The infiltration of CD66b-positive cells was semi-quantitatively evaluated along the tumor invasive front, in the tumor center, and within the tumor epithelium (intraepithelial expression). We found that poor infiltration of CD66b-positive cells in the tumor front indicated a worse patient prognosis. The prognostic significance of CD66b infiltration was found to be mainly independent of tumor molecular characteristics and maintained significance in multivariable analysis of stage I-II colon cancers. We further analyzed the prognostic impact of CD66b-positive cells in relation to other immune markers (NOS2, CD163, Tbet, FOXP3, and CD8) and found that neutrophil infiltration, even though strongly correlated to infiltration of other immune cell subsets, had additional prognostic value. In conclusion, we find that low infiltration of neutrophils in the tumor front is an independent prognostic factor for a poorer patient prognosis in early stages of colon cancers. Further studies are needed to elucidate the biological role of neutrophils in colorectal carcinogenesis.

TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer.

The anti-tumor immune response has been shown to be of great prognostic importance in colorectal cancer (CRC) and so has the tumors ability for immune evasion. Our aim of this study was to investigate tumor factors that influence immunity. We used a gene expression array to search for potential mechanisms of tumor immune escape. One candidate gene identified was TAP1, involved in antigen presentation by MHC class I. TAP1 protein expression was evaluated by immunohistochemistry in 436 CRC patients of the Colorectal Cancer in Umeå Study cohort. We found a significant association between a downregulated expression of TAP1 and low infiltration of various subtypes of lymphocytes as well as macrophages. A downregulated expression of TAP1 was further found to be independent of molecular characteristics, suggesting TAP1 down-regulation to reach beyond the well described highly immunogenic MSI CRCs. A low expression of TAP1 was also significantly associated with poor prognosis in patients with CRC, a result that stayed significant in tumor front of early stage tumors (stage I-II) through multivariable analyses. Furthermore, we found that TAP1 expression was inversely correlated with methylation at sites in close proximity to the promoter region. In summary, our results show down-regulation of TAP1 to be a general mechanism of tumor immune escape in CRC and a poor prognostic factor in stage I-II CRC patients. We also suggest that methylation of the TAP1 gene may be a putative mechanism for TAP1 downregulation.

The infiltration, and prognostic importance, of Th1 lymphocytes vary in molecular subgroups of colorectal cancer.

Giving strong prognostic information, T-cell infiltration is on the verge of becoming an additional component in the routine clinical setting for classification of colorectal cancer (CRC). With a view to further improving the tools for prognostic evaluation, we have studied how Th1 lymphocyte infiltration correlates with prognosis not only by quantity, but also by subsite, within CRCs with different molecular characteristics (microsatellite instability, CpG island methylator phenotype status, and BRAF and KRAS mutational status). We evaluated the Th1 marker T-bet by immunohistochemistry in 418 archival tumour tissue samples from patients who underwent surgical resection for CRC. We found that a high number of infiltrating Th1 lymphocytes is strongly associated with an improved prognosis in patients with CRC, irrespective of intratumoural subsite, and that both extent of infiltration and patient outcome differ according to molecular subgroup. In brief, microsatellite instability, CpG island methylator phenotype-high and BRAF mutated tumours showed increased infiltration of Th1 lymphocytes, and the most pronounced prognostic effect of Th1 infiltration was found in these tumours. Interestingly, BRAF mutated tumours were found to be more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours whereas the opposite was seen for KRAS mutated tumours. These differences could be explained at least partly by our finding that BRAF mutated, in contrast to KRAS mutated, CRC cell lines and tumour specimens expressed higher levels of the Th1-attracting chemokine CXCL10, and reduced levels of CCL22 and TGFB1, stimulating Th2/Treg recruitment and polarisation. In conclusion, the strong prognostic importance of Th1 lymphocyte infiltration in CRC was found at all subsites evaluated, and it remained significant in multivariable analyses, indicating that T-bet may be a valuable marker in the clinical setting. Our results also indicate that T-bet is of value when analysed in molecular subgroups of CRC, allowing identification of patients with especially poor prognosis who are in need of extended treatment.

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.

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.

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.

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.

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.