Roland S. Croner

TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

BackgroundHistological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited.MethodsTissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fishers exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed.ResultsHigh-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2 expression was an independent prognostic factor for cancer related survival.ConclusionHistological phenotype and clinical behaviour of colon cancer is not only influenced by mutational incidents in tumour cells but also affected by interaction of tumour tissue with inflammatory cells like macrophages and associated stroma and TGF-beta signalling is one important part of this crosstalk. Further studies are needed to elucidate the exact mechanisms.

Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine

Pseudouni- or multiaxonal Dogiel type II neurons are the intrinsic primary afferent (sensory) neurons (IPANs) in the guinea pig small intestine. Our aim was to decipher the chemical code of human myenteric type II neurons and to establish their putative vertical projections, i.e., from the myenteric plexus to the submucosa/mucosa. Additionally, we tried to distinguish them chemically from uniaxonal, dendritic type V neurons displaying, at first glance, similar shapes, i.e., smoothly contoured cell bodies with several long processes. Wholemount preparations of the myenteric plexus were immunohistochemically double or triple stained for neurofilaments (NF) and one or two of the following peptides: calbindin, calretinin (CR), calcitonin gene-related peptide (CGRP), somatostatin (SOM) and substance P (SP). In each triple stained wholemount three counts were conducted: (1) NF-positive pseudouni- or multiaxonal (type II) neurons including their reactivities for the above peptides, (2) uniaxonal or NF-negative neurons displaying coreactivities for the above peptides and (3) NF-reactive type V neurons taking into account their reactivities for the above markers. Additionally, type II neurons, which had an axon leading into (disrupted) interconnecting strands towards the submucosa were counted and somal areas of types II and V neurons were measured. The majority of myenteric type II neurons displayed coreactivities for SOM/CR (89.6%), SOM/SP (86.6%) and SP/CR (81.6%), respectively. A minority of type II neurons was positive for CGRP or calbindin. A small population with type III morphology (uniaxonal, long and slender dendrites) displayed the same coreactivities as type II neurons. In contrast, not one single type V neuron was coreactive for SOM/CR, SOM/SP or SP/CR. Out of 627 type II neurons counted in six wholemounts, 84 type II neurons displayed an axon which could be followed into disrupted interconnecting strands indicating a vertical projection pattern. Somal areas of type II neurons were twice as big as those of type V neurons (904+/-210 versus 449+/-110 microm(2)). In conclusion, most human myenteric type II neurons contain SOM, SP and CR. We suggest they are the human IPANs. Type V neurons are both morphologically and chemically distinctly different from type II neurons and may represent descending interneurons. Further studies have to decipher the type-specific chemical code of type II neurons distinguishing them also from type III neurons.

Angiostatic immune reaction in colorectal carcinoma: Impact on survival and perspectives for antiangiogenic therapy

Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein‐1 (GBP‐1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP‐1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP‐1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as determined by immunohistochemical investigation of 388 sporadic CRC. Cancer‐related 5‐year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP‐1‐positive CRC. Multivariate analysis showed that GBP‐1 is an independent prognostic factor indicating a reduction of the relative risk of cancer‐related death by the half (p = 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP‐1‐positive (n = 12) and ‐negative (n = 12) tumors showed that particularly IFN‐γ‐induced genes including the major antiangiogenic chemokines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP‐1. Altogether our findings indicated that GBP‐1 may be a novel biomarker and an active component of a Th‐1‐like angiostatic immune reaction in CRC. This reaction may affect patients response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induction of a Th‐1‐like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC.

Hepatic platelet and leukocyte adherence during endotoxemia

IntroductionLiver microcirculation disturbances are a cause of hepatic failure in sepsis. Increased leukocyte-endothelial interaction, platelet adherence and impaired microperfusion cause hepatocellular damage. The time course and reciprocal influences of ongoing microcirculatory events during endotoxemia have not been clarified.MethodsMale Wistar rats (232 ± 17 g) underwent cecal ligation and puncture (CLP). Intravital microscopy (IVM) was performed 0, 1, 3, 5, 10 and 20 hours after CLP. Mean erythrocyte velocity, leukocyte and platelet rolling in postsinusoidal venules and sticking of leukocytes and platelets in postsinusoidal venules and hepatic sinusoids were determined. Heart rate (HR), mean arterial pressure (MAP) and portal venous blood flow (PBF) were measured. Blood count and investigation of hepatic enzyme release was performed after each IVM time point.ResultsHepatic platelet-endothelial adherence in liver sinusoids and postsinusoidal venules occurred one hour after the induction of endotoxemia. Leukocyte-endothelial interaction started three to five hours after CLP. A decrease of hepatic microperfusion could be observed at three hours in sinusoids and ten hours in postsinusoidal venules after CLP, although PBF was reduced one hour after CLP. HR remained stable and MAP decreased ten hours after CLP. Hepatic enzymes in blood were significantly elevated ten hours after CLP.ConclusionHepatic platelet-endothelial interaction is an early event during endotoxemia. Leukocyte adherence occurs later, which underlines the probable involvement of platelets in leukocyte recruitment. Although PBF is reduced immediately after CLP, the later onset of hepatic microperfusion decrease makes the existence of autoregulatory liver mechanisms likely.

Multivisceral resection for colon carcinoma.

PURPOSE: The aim of curative surgery for colon carcinoma is the complete resection of the neoplasm. In locally advanced colon carcinomas with adhesion to neighboring organs, standard surgical procedures often turn into multivisceral resections. The purpose of this study was to investigate the value of multivisceral resection in primary colon carcinomas and factors influencing its success. METHODS: Prospectively collected data for 174 patients from the Erlangen Registry for Colorectal Carcinomas who underwent multivisceral resection for colon carcinoma from 1978 through 2002 were analyzed. Multivisceral resection was defined as the excision or resection of at least one further organ in addition to the carcinoma-affected colon. Postoperative complications, locoregional tumor recurrence, distant metastases, and cancer-related survival were evaluated after a five-year follow-up. RESULTS: Multivisceral resection most commonly involved parts of the small intestine (31.6%), urinary bladder (27.0%), and the abdominal wall (15.5%). R0 resection (no residual tumor) was achieved in 93.1%. Overall, postoperative complications occurred in 25.8%, and the postoperative mortality rate was 6.9%. For patients with R0 resection, the Kaplan-Meier estimate of five-year cancer-related survival was 80.7%; no patient with R1 or R2 resection survived for 5 years. The five-year rate of locoregional tumor recurrence was 6.5%, and the five-year rate of distant metastases was 24.2%. The presence of lymphatic metastases was a significant prognostic factor for locoregional tumor recurrence, distant metastases, and cancer-related survival. CONCLUSION: The high percentage of R0 resections achieved through multivisceral resection justifies this procedure for locally advanced colon carcinomas and highlights the importance of experienced, well-trained surgeons to decrease the incidence of locoregional recurrence.

Notch3 signalling promotes tumour growth in colorectal cancer

Increased Notch1 activity has been observed in intestinal tumours, partially accomplished by β‐catenin‐mediated up‐regulation of the Notch ligand Jagged‐1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved is unclear. Microarray data indicated that Notch3 transcript levels are significantly up‐regulated in primary and metastatic CRC samples compared to normal mucosa. Moreover, Notch3 protein was expressed at strong/moderate levels by 19.7% of 158 CRC samples analysed, and at weak levels by 51.2% of the samples. Intrigued by these findings, we sought to investigate whether Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumourigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including Notch3, Delta‐like 4 (DLL4), and Jagged‐1 ligands. Stimulation with immobilized recombinant DLL4 or transduction with DLL4‐expressing vectors dramatically increased Notch3 expression in CRC cells, associated with accelerated tumour growth. Forced expression of an active form of Notch3 mirrored the effects of DLL4 stimulation and increased tumour formation. Conversely, attenuation of Notch3 levels by shRNA resulted in perturbation of the cell cycle followed by reduction in cell proliferation, clonogenic capacity, and inhibition of tumour growth. Altogether, these findings indicate that Notch3 can modulate the tumourigenic properties of CRC cells and contributes to sustained Notch activity in DLL4‐expressing tumours. Copyright

DNA Stool Test for Colorectal Cancer: Hypermethylation of the Secreted Frizzled-Related Protein-1 Gene

PurposeTo investigate a potential mode of noninvasive screening for colorectal cancer, we evaluated the hypermethylation of the secreted frizzled-related protein-1 gene promoter in human stool DNA.MethodsIn stool samples from 36 patients with colorectal neoplasia (7 adenoma, 29 colorectal cancer) and 17 healthy control subjects, isolated DNA was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction with primers specific for methylated or unmethylated promoter sequences of the secreted frizzled-related protein-1 gene.ResultsHypermethylation of the secreted frizzled-related protein-1 promoter was present in the stool DNA of patients with adenoma and colorectal cancer. A sensitivity of 89 percent and specificity of 86 percent were achieved in the detection of colorectal neoplasia. The difference in hypermethylation status of the secreted frizzled-related protein-1 promoter between the patients with colorectal neoplasia and the control group was statistically highly significant (P < 0.001). Adenoma and early tumor Stage I (International Union Against Cancer) displayed both unmethylated and methylated secreted frizzled-related protein-1 promoter sequences, whereas advanced tumor stages showed only methylated secreted frizzled-related protein-1 (P = 0.05).ConclusionsThe results indicate that this DNA stool test of hypermethylation of the secreted frizzled-related protein-1 promoter is a sensitive and specific method. It has the potential of a clinically useful test for the early detection of colorectal cancer.

Common denominator genes that distinguish colorectal carcinoma from normal mucosa

PurposeMicroarray technology has been used by a growing number of investigators and several studies have been published that list hundreds of genes differentially expressed by colorectal carcinoma (CRC) and normal mucosa (MC). On the basis of our own and other investigators’ microarray data, our goal was to identify a common denominator gene cluster distinguishing CRC from MC.MethodsThirty GeneChips (HG-U133A, Affymetrix) were hybridized, 20 with RNA of CRC stages I–IV (UICC) and 10 with MC. Expression signals showing at least a 4-fold difference between CRC and MC (p<0.01) were identified as differentially expressed. In addition, in our integrative data analysis approach only those genes whose expression was altered simultaneously in at least 2 of 5 recently published studies were subjected to an unsupervised hierarchical cluster analysis.ResultsWe detected 168 up- and 283 down-regulated genes in CRC relative to MC. Twenty-three genes were filtered from the five articles reviewed. An unsupervised hierarchical cluster analysis of these 23 genes confirmed the high specificity of these genes to differentiate between CRC and MC in our microarray data.ConclusionsColorectal cancer and mucosa could be clearly separated by 23 genes selected for being differentially expressed more than once in a recent literature review. These genes represent a common denominator gene cluster that can be used to distinguish colorectal MC from CRC.

Age and manifestation related symptoms in familial adenomatous polyposis

BackgroundTo identify early symptoms of familial adenomatous polyposis with a view to improve early diagnosis and treatment. Diagnosis on the basis of genetic testing is usually limited to where there is a known family history, so FAP is more usually diagnosed on clinical grounds. Except for those identified via FAP registers, the majority of patients are symptomatic at the time of diagnosis.MethodsWe undertook a retrospective study of 143 FAP patients treated at the Department of Surgery, University of Erlangen between 1971 and 2000. We identified patterns of symptoms, endoscopic findings and extracolonic manifestations in three age groups.ResultsFAP was diagnosed clinically on the basis of symptoms in 84% (120/143) of these patients. Most presented with intestinal symptoms such as colonic bleeding (68%) and diarrhea (42%). All but one of the patients between 20 and 40 years old had rectal polyps (98.7%, 75/76), whereas in those over 40 years old the prevalence was 76% (35/46). Non-specific symptoms such as abdominal pain, fatigue and bloating were less frequent and were mainly reported by patients older than 40.ConclusionThe commonest presenting features of FAP are alteration of bowel habit and rectal bleeding, but both are found in many other conditions. Patients with these findings need immediate endoscopy to allow prompt diagnosis and prophylactic surgery.

GBP-1 acts as a tumor suppressor in colorectal cancer cells.

The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.