Diarmuid P. O'Donoghue

Tumor budding is a strong and reproducible prognostic marker in T3N0 colorectal cancer.

Background Tumor budding along the advancing front of colorectal adenocarcinoma is an early event in the metastatic process. A reproducible, prognostic budding scoring system based on outcomes in early stage colorectal cancer has not been established. Design One hundred twenty-eight T3N0M0 colorectal carcinoma patients with known outcome were identified. Tumor budding was defined as isolated tumor cells or clusters of <5 cells at the invasive tumor front. Tumor bud counts were generated in 5 regions at 200× by 2 pathologists (conventional bud count method). The median bud count per case was used to divide cases into low (median=0) and high budding (median ≥1) groups. Forty cases were reevaluated to assess reproducibility using the conventional and a novel rapid bud count method. Results Fifty-seven (45%) carcinomas had high and 71 (55%) had low budding scores. High budding was associated with an infiltrative growth pattern (P<0.0001) and lymphovascular invasion (P=0.005). Five-year cancer-specific survival was significantly poorer in high compared with low budding groups: 63% versus 91%, respectively, P<0.0001. Multivariate analysis demonstrated tumor budding to be independently prognostic (hazard ratio=4.76, P<0.001). Interobserver agreement was at least equivalent comparing the conventional to the rapid bud count methods: 87.5% agreement (κ=0.75) versus 92.5% agreement (κ=0.85), respectively. Conclusions Tumor budding is a strong, reproducible, and independent prognostic marker of outcome that is easily assessed on hematoxylin and eosin slides. This may be useful for identifying the subset of T3N0M0 patients at high risk of recurrence who may benefit from adjuvant therapy.

Association of NOD2 with Crohn's disease in a homogenous Irish population.

Linkage of IBD to the pericentromeric region of chromosome 16 has been widely confirmed by analyses of multiple populations. The NOD2 gene is located in the peak region of linkage on chromosome 16 and thought to be involved in the activation of nuclear factor (NF) κB in response to bacterial components. Mutations in the NOD2 gene are found to be strongly associated with susceptibility to Crohns disease (CD). A total of 65 Irish CD families were genotyped to determine if NOD2 mutations conferred susceptibility to CD and the prevalence of these mutations in sporadic and familial forms of the disease. The Irish population is relatively homogenous and thus may provide advantages in genetic studies of complex diseases. We confirmed the IBD1 locus as a susceptibility locus for IBD within the Irish population by linkage analysis followed by linkage disequilibrium studies. No significant evidence of linkage was observed to the previously identified regions on chromosomes 1, 12 and 14. In all, 131 CD affected families were then genotyped for seven of the previously published NOD2 single-nucleotide polymorphisms (SNPs). Allelic transmission distortion was investigated using the pedigree disequilibrium test (PDT). SNP13 (3020insC) was found to be associated with CD (P=0.0186). Patients who possessed a rare allele of SNP8, 12 or 13 presented earlier when compared to patients without rare variants (mean age, 20.1 vs 24 years, P=0.011) and the rare allele of SNP13 was observed to be predominantly linked to ileal disease (P=0.02). This report confirms the importance of NOD2 as a susceptibility gene for CD within the Irish population.

Tumour Tissue Microenvironment Can Inhibit Dendritic Cell Maturation in Colorectal Cancer

Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

Long-term clinical results of ileocecal resection for Crohn's disease.

Background: The efficacy of biologic agents in Crohns disease (CD) has led to proposals that they be introduced early in the disease (top‐down treatment) with the aim of reducing corticosteroid dependency and surgical resection. However, the long‐term use of biologic agents in limited CD may be difficult to justify. The aims were to assess outcomes for ileocecal resection in CD and evaluate its role in the current era. Methods: The study included 139 CD patients who underwent ileocecal resection between 1980 and 2000. Data were retrieved from a prospectively maintained database. Disease recurrence was defined as symptoms in addition to endoscopic or radiological evidence of disease activity. Severe disease recurrence was defined as a need for repeat resection surgery. Results: Seventy‐two (52%) patients developed disease recurrence. Median (interquartile range) time to recurrence was 7.1 (5–10.6) years. Forty‐nine (35%) patients required repeat resection surgery. Median (IQ range) time to repeat surgery was 7.2 (4.9–10.8) years. The presence of granulomas was associated with disease recurrence (P = 0.03) and repeat resection surgery (P = 0.01). Conclusions: Long‐term outcomes for ileocecal resection in CD are excellent with 48% of patients remaining symptom‐free and only 35% requiring repeat resection surgery at 10 years. This should be borne in mind when considering biologic therapy.

Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer

Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro‐ and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro‐Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

Neurokinin‐1 receptor (NK‐1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P

We have previously shown that the receptor for substance P (SP), neurokinin‐1 receptor (NK‐1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK‐1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK‐1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK‐1R mRNA, and immunohistochemistry to detect NK‐1R protein, colonic epithelial cells were found to express NK‐1R in vivo. In contrast, colon epithelial cell lines (Caco‐2, HT29, SW620, T84) were negative for NK‐1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN‐γ, TNF‐α, and IL‐1β, caused induction of NK‐1R expression. Expression of NK‐1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine‐treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10−10–10−8 M) elicited a dose‐dependent proliferative effect on cytokine‐stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine‐induced NK‐1R, since an NK‐1R‐specific antagonist (Spantide 1) completely blocked SP‐mediated proliferation in the cytokine‐treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK‐1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine‐induced NK‐1R. J. Cell. Physiol. 197: 30–41, 2003© 2003 Wiley‐Liss, Inc.

Efficacy of Adalimumab as a long term maintenance therapy in ulcerative colitis.

INTRODUCTION Adalimumab is a recombinant human IgG1 monoclonal antibody to TNF-alpha. There are limited data with regard to its efficacy in ulcerative colitis. We report experience of adalimumab in ulcerative colitis in a single centre with a focus on the ability of this agent to maintain response and avoid colectomy in the medium to long-term. METHODS Twenty-three ulcerative colitis patients (mean age 32 years; 7 female) who received adalimumab were identified from a prospectively maintained database of over 2700 IBD patients. The primary study endpoint was treatment failure defined as discontinuation of adalimumab due to lack of efficacy, as defined by requiring an alternative maintenance therapy or colectomy, or intolerance. Colectomy rate was recorded as a secondary endpoint. RESULTS Most patients (96%) had received immunosuppressants prior to adalimumab therapy (infliximab 20/23 87%). Sixteen of 23 patients (70%) discontinued adalimumab. Six primary failures, 8 secondary loss of response, one had unacceptable side effects and one discontinued treatment after 6 months but remains in remission. Overall estimated cumulative treatment failure rates at 6, 12 and 24 months were 50%, 65% and 72% respectively. Median follow-up in patients continuing adalimumab is 23 months (IQR 17-31 months). Treatment failure was unrelated to patient age, gender, disease extent, smoking status or CRP. Colectomy free survival was 59% at 2 years. No patient experienced a major adverse event. CONCLUSION Adalimumab shows some efficacy as a maintenance strategy in Ulcerative Colitis, but only a limited proportion of patients remain well on continued treatment at 2 years.

Radiation and chemotherapy bystander effects induce early genomic instability events: telomere shortening and bridge formation coupled with mitochondrial dysfunction.

The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapy-treated explant tissues. Bystander cells exposed to media from 2Gy, 5Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0Gy) at 24h. There was no significant difference between 2Gy and 5Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p=0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p=0.02) and mitochondrial membrane potential increased (p=0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial function through overexpression of MnSOD significantly rescues nuclear instability events; anaphase bridges and telomere length shortening.

High Clusterin Expression Correlates with a Poor Outcome in Stage II Colorectal Cancers

The role of clusterin in tumor growth and progression remains unclear. Overexpression of cytoplasmic clusterin has been studied in aggressive colon tumors; however, no correlation between clusterin expression and survival in colorectal cancer has been identified to date. We assessed levels of clusterin expression in a group of stage II colorectal cancer patients to assess its utility as a prognostic marker. The study included 251 patients with stage II colorectal cancer. Tissue microarrays were constructed and immunohistochemistry done and correlated with clinical features and long term outcome. Dual immunofluorescence and confocal microscopy were used with terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling probes and clusterin antibody to assess the degree of co localization. Percentage epithelial cytoplasmic staining was higher in tumor compared with nonadjacent normal mucosa (P < 0.001). Within the stromal compartment, percentage cytoplamic staining and intensity was lower in tumor tissue compared with normal nonadjacent mucosa (P ≤ 0.001). Survival was significantly associated with percentage epithelial cytoplasmic staining (P < 0.001), epithelial cytoplasmic staining intensity (P < 0.001), percentage stromal cytoplasmic staining (P = 0.002), and stromal cytoplasmic staining intensity (P < 0.001). Clusterin levels are associated with poor survival in stage II colorectal cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(2):393–9)

CD4+CD8+ and CD8α+β- T lymphocytes in human small intestinal lamina propria

Objective To examine CD8 expression by T-lymphocyte subpopulations from disease-free human lamina propria. Methods Single-cell suspensions were prepared from the epithelial layer and the lamina propria of small intestinal biopsies obtained endoscopically from disease-free patients. Monoclonal antibodies against CD3, CD4, CD8, CD56, CD8αβ, CD8α, TCRαβ and TCR-γ&dgr; were used for dual and three-colour flow cytometric analysis. Results In addition to classical CD4+ and CD8+ T lymphocytes a substantial proportion of lamina propria T lymphocytes were CD4+CD8+ or ‘double positive’ (mean 14%, range 4–26%). This population was significantly lower in the epithelial layer of the same patients (mean 7%, range 3–21 %, P < 0.007). Three-colour flow cytometric analysis revealed that expression of the CD8 molecule on double positive T cells in the lamina propria was limited to the CD8α chain. Furthermore, of the CD8+ population, CD8+ T cells which only expressed the a chain were present in greater numbers in the lamina propria (mean 35%, range 14–54%) than in the epithelial layer (mean 18%, range 5–37%, P < 0.02). NK (CD56+) cells were not detected and few γ&dgr;TCR+ T lymphocytes were detected in the lamina propria (mean 2%, range 0.5–6.6%) when compared with the epithelial layer (mean 8%, range 0.2–14%, P < 0.008). Conclusion A significant population of CD4+CD8α+ T lymphocytes which are CD8β chain negative have been detected in the intestinal lamina propria. These cells form a more significant component of the lamina propria than the epithelial layer T-cell repertoire and may have a unique function in intestinal immunoregulation.