Anna M. Dahlin

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 role of the CpG island methylator phenotype in colorectal cancer prognosis depends on microsatellite instability screening status

Purpose: The aim of this study was to relate the CpG island methylator phenotype (CIMP; characterized by extensive promoter hypermethylation) to cancer-specific survival in colorectal cancer, taking into consideration relevant clinicopathologic factors, such as microsatellite instability (MSI) screening status and the BRAF V600E mutation. Experimental Design: Archival tumor samples from 190 patients from the Northern Sweden Health and Disease Study (NSHDS) and 414 patients from the Colorectal Cancer in Umeå Study (CRUMS), including 574 with cancer-specific survival data, were analyzed for an eight-gene CIMP panel using quantitative real-time PCR (MethyLight). MSI screening status was assessed by immunohistochemistry. Results: CIMP-low patients had a shorter cancer-specific survival compared with CIMP-negative patients (multivariate hazard ratio in NSHDS, 2.01; 95% confidence interval, 1.20-3.37; multivariate hazard ratio in CRUMS, 1.48; 95% confidence interval, 1.00-2.22). This result was similar in subgroups based on MSI screening status and was statistically significant in microsatellite stable (MSS) tumors in NSHDS. For CIMP-high patients, a shorter cancer-specific survival compared with CIMP-negative patients was observed in the MSS subgroup. Statistical significance was lost after adjusting for the BRAF mutation, but the main findings were generally unaffected. Conclusions: In this study, we found a poor prognosis in CIMP-low patients regardless of MSI screening status, and in CIMP-high patients with MSS. Although not consistently statistically significant, these results were consistent in two separate patient groups and emphasize the potential importance of CIMP and MSI status in colorectal cancer research. Clin Cancer Res; 16(6); 1845–55

Colorectal cancer prognosis depends on T-cell infiltration and molecular characteristics of the tumor.

The aim of this study was to relate the density of tumor infiltrating T cells to cancer-specific survival in colorectal cancer, taking into consideration the CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) screening status. The T-cell marker CD3 was stained by immunohistochemistry in 484 archival tumor tissue samples. T-cell density was semiquantitatively estimated and scored 1–4 in the tumor front and center (T cells in stroma), and intraepithelially (T cells infiltrating tumor cell nests). Total CD3 score was calculated as the sum of the three CD3 scores (range 3–12). MSI screening status was assessed by immunohistochemistry. CIMP status was determined by quantitative real-time PCR (MethyLight) using an eight-gene panel. We found that patients whose tumors were highly infiltrated by T cells (total CD3 score ≥7) had longer survival compared with patients with poorly infiltrated tumors (total CD3 score ≤4). This finding was statistically significant in multivariate analyses (multivariate hazard ratio, 0.57; 95% confidence interval, 0.31–1.00). Importantly, the finding was consistent in rectal cancer patients treated with preoperative radiotherapy. Although microsatellite unstable tumor patients are generally considered to have better prognosis, we found no difference in survival between microsatellite unstable and microsatellite stable (MSS) colorectal cancer patients with similar total CD3 scores. Patients with MSS tumors highly infiltrated by T cells had better prognosis compared with intermediately or poorly infiltrated microsatellite unstable tumors (log rank P=0.013). Regarding CIMP status, CIMP-low was associated with particularly poor prognosis in patients with poorly infiltrated tumors (multivariate hazard ratio for CIMP-low versus CIMP-negative, 3.07; 95% confidence interval, 1.53–6.15). However, some subset analyses suffered from low power and are in need of confirmation by independent studies. In conclusion, patients whose tumors are highly infiltrated by T cells have a beneficial prognosis, regardless of MSI, whereas the role of CIMP status in this context is less clear.

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.

Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion.

Cancer-associated fibroblasts expressing fibroblast activation protein (FAP) have been implicated in the invasive behavior of colorectal cancer. In this study, we use FAP expression as a marker of fibroblast activation and analyze the effect of activated fibroblasts on colorectal cancer migration and invasion in experimental cell studies. We also investigated the expression pattern of FAP in cancer-associated fibroblasts during transformation from benign to malignant colorectal tumors. In immunohistochemical analyses, FAP was expressed in fibroblasts in all colorectal cancer samples examined, whereas all normal colon, hyperplastic polyps, or adenoma samples were negative. In in vitro studies, conditioned medium from colon cancer cells, but not adenoma cells, activated fibroblasts by inducing FAP expression. These activated fibroblasts increased the migration and invasion of colon cancer cells in Boyden chamber experiments and in a three-dimensional cell culture model. We identify fibroblast growth factor 1/fibroblast growth factor receptor 3 (FGF1/FGFR-3) signaling as mediators leading to the increased migration and invasion. Activated fibroblasts increase their expression of FGF1, and by adding a fibroblast growth factor receptor inhibitor, as well as an FGF1-neutralizing antibody, we reduced the migration of colon cancer cells. Our findings provide evidence of a possible molecular mechanism involved in the cross talk between cancer cells and fibroblasts leading to cancer cell invasion.

High tumour cannabinoid CB1 receptor immunoreactivity negatively impacts disease-specific survival in stage II microsatellite stable colorectal cancer.

Background There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB1 receptor immunoreactive intensity (CB1IR intensity) is associated with disease severity and outcome. Methodology/Principal Findings CB1IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB1IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB1IR <2 and ≥2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB1IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB1IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB1IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB1IR upon disease specific survival. The 5 year probabilities of event-free survival were: 85±5 and 66±8%; tumour interior, 86±4% and 63±8% for the CB1IR<2 and CB1IR≥2 groups, respectively. Conclusions/Significance The level of CB1 receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB1IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients.

One-carbon metabolism and CpG island methylator phenotype status in incident colorectal cancer : a nested case-referent study

ObjectiveWe related prediagnostic plasma folate, vitamin B12, and total homocysteine concentrations, and the MTHFR 677C>T and 1298A>C polymorphisms, to the risk of colorectal cancer with and without the CpG island methylator phenotype (CIMP).MethodsThis was a nested case–referent study of 190 cases and double, matched referents from the large, population-based Northern Sweden Health and Disease Study. Using archival tumor tissue, promoter methylation in an eight-gene panel was analyzed by MethyLight.ResultsA reduced risk of CIMP-low/CIMP-high CRC (≥1 gene methylated) was observed in subjects with very low plasma folate concentrations [multivariate odds ratio 2.96 (95% CI 1.24–7.08) for quintiles two to five versus one (lowest)]. With the exception of a reduced risk in MTHFR 677 TT-homozygotes, none of the other one-carbon variables were associated with the risk of CIMP-low/CIMP-high CRC. For CIMP-negative CRC, only the MTHFR polymorphisms were statistically significantly related to risk, inversely for 677C>T and positively for 1298A>C, but a tendency toward a reduced risk was observed in subjects with an adequate methyl availability, combining the plasma variables [multivariate odds ratio 0.61 (95% CI 0.32–1.15)].ConclusionThough limited by low power, these findings suggest the possibility of different roles for one-carbon metabolism in different pathways of colorectal tumorigenesis.

Plasma vitamin B12 concentrations and the risk of colorectal cancer: a nested case-referent study.

In this nested case‐referent study, we related plasma concentrations of vitamin B12 to the risk of colorectal cancer, taking into consideration prediagnostic plasma folate and total homocysteine concentrations. Subjects were 226 cases and double matched referents from the population‐based Northern Sweden Health and Disease Study. Follow‐up times from recruitment to diagnosis ranged from 0.1 to 12.7 years, with a median of 4.2 years. Plasma vitamin B12 concentrations were inversely associated with the risk of rectal cancer: univariate odds ratio for the highest versus lowest quintile 0.34 (95% confidence interval (95% CI) 0.13–0.83), ptrend = 0.004. Risk estimates were attenuated slightly but remained statistically significant after adjustment for body mass index, current smoking, recreational and occupational physical activity, alcohol intake and prediagnostic plasma folate and total homocysteine concentrations: OR 0.30 (95% CI 0.08–0.99), ptrend = 0.025. The corresponding univariate and fully adjusted odds ratios for colon cancer were 1.25 (CI 0.66–2.36), ptrend = 0.185 and 1.42 (CI 0.67–3.05), ptrend = 0.113, respectively. The observed over‐risk was attributable to left‐sided colon cancer. Interaction analyses including vitamin B12, folate and homocysteine were in line with the results for vitamin B12 alone. In conclusion, these results suggest that increasing levels of plasma vitamin B12, alone or together with other factors involved in one‐carbon metabolism, may reduce the risk of rectal cancer, whereas for colon cancer, the association appears to be less clear.

High SMAD4 levels appear in microsatellite instability and hypermethylated colon cancers, and indicate a better prognosis

Colorectal cancer (CRC) is one of the most common causes of cancer‐related deaths in western countries. CRC are commonly divided in cancers showing microsatellite stability (MSS) or microsatellite instability (MSI). A more novel classification is dependent on promoter hypermethylation of CpG islands (the CpG island methylator phenotype, CIMP), where cancers show high, low or negative methylation status. SMAD4, located on chromosome 18q, has been thoroughly investigated during the last years. Loss of SMAD4 expression has been reported to correlate with poor CRC patient prognosis. In this study, we analyze the impact of SMAD4 expression on prognosis in relation to MSI screening status and CIMP status. Four hundred and seventy‐nine paraffin‐embedded specimens of CRC were examined for nuclear SMAD4 expression using immunohistochemistry. The tumors were scored loss (−), moderate (+) and high (++) expressing tumors. Loss of SMAD4 correlated significantly with decreased survival in all colon cancer patients. High SMAD4 expression, however, was significantly associated with increased survival, especially in colon cancer patients, which has undergone potential curative surgery. In addition, in MSI tumors and CIMP‐high tumors, high SMAD4 expression was significantly related to increase in survival, while loss of SMAD4 resulted in a significantly poorer prognosis. SMAD4 expression was not correlated to prognosis in rectal cancer cases. We conclude, loss of SMAD4 indicates a poor prognosis in colon cancer patients. The novel findings that high SMAD4 expression predicts a better prognosis suggests that SMAD4 immunohistochemistry could constitute a prognostic marker in combination with CIMP and MSI screening status.

Known glioma risk loci are associated with glioma with a family history of brain tumours -- a case-control gene association study.

Familial cancer can be used to leverage genetic association studies. Recent genome‐wide association studies have reported independent associations between seven single nucleotide polymorphisms (SNPs) and risk of glioma. The aim of this study was to investigate whether glioma cases with a positive family history of brain tumours, defined as having at least one first‐ or second‐degree relative with a history of brain tumour, are associated with known glioma risk loci. One thousand four hundred and thirty‐one glioma cases and 2,868 cancer‐free controls were identified from four case–control studies and two prospective cohorts from USA, Sweden and Denmark and genotyped for seven SNPs previously reported to be associated with glioma risk in case–control designed studies. Odds ratios were calculated by unconditional logistic regression. In analyses including glioma cases with a family history of brain tumours (n = 104) and control subjects free of glioma at baseline, three of seven SNPs were associated with glioma risk: rs2736100 (5p15.33, TERT), rs4977756 (9p21.3, CDKN2A‐CDKN2B) and rs6010620 (20q13.33, RTEL1). After Bonferroni correction for multiple comparisons, only one marker was statistically significantly associated with glioma risk, rs6010620 (ORtrend for the minor (A) allele, 0.39; 95% CI: 0.25–0.61; Bonferroni adjusted ptrend, 1.7 × 10−4). In conclusion, as previously shown for glioma regardless of family history of brain tumours, rs6010620 (RTEL1) was associated with an increased risk of glioma when restricting to cases with family history of brain tumours. These findings require confirmation in further studies with a larger number of glioma cases with a family history of brain tumours.