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author:("remmele, P")
1.  Multi-colour FISH in oesophageal adenocarcinoma—predictors of prognosis independent of stage and grade 
British Journal of Cancer  2014;110(12):2985-2995.
Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour Fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples.
To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data.
The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months.
Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.
PMCID: PMC4056055  PMID: 24853183
multi-colour FISH; oesophageal adenocarcinoma; ERBB2; ZNF217; MYC; CDKN2A
2.  Morphological characterisation of Crohn’s disease fistulae 
Gut  2004;53(9):1314-1321.
Background: Fistulae are a common complication in up to 35% of all patients with Crohn’s disease. Their therapy is difficult and frequently unsatisfactory. To date, no histological comparison of Crohn’s disease fistulae with non-inflammatory bowel disease fistulae has been performed. In addition, Crohn’s disease fistulae have not been well characterised morphologically.
Methods: Eighty four fistulae from Crohn’s disease patients were compared with 13 fistulae from controls. Haematoxylin-eosin staining, electron microscopy, and immunohistochemistry for panCytokeratin (epithelial cells), CD20 (B cells), CD45R0 (T cells), and CD68 (macrophages) were performed according to standard techniques. In addition, histopathological findings were compared with clinical and laboratory data.
Results: In 31.0% of controls and 27.4% of Crohn’s disease specimens, fistulae had a lining of flattened intestinal epithelium without goblet cells or, in the case of cutaneous/perianal disease, narrow squamous epithelium. Non-epithelialised fistulae were covered by a thin layer of (myo)fibroblasts, focally forming a new basement membrane, as demonstrated by electron microscopy. All fistulae were surrounded by granulation tissue. Crohn’s disease fistulae presented with central infiltration by CD45R0+ T cells, followed by a small band of CD68+ macrophages and dense accumulation of CD20+ B cells. In contrast, in controls, there was dense infiltration by CD68+ macrophages with only few CD20+ B cells and CD45R0+ T lymphocytes.
Conclusions: Fistulae in Crohn’s disease differ markedly from non-Crohn’s disease fistulae with regard to their cellular composition. The presence of an epithelial lining in a subgroup of fistulae may be important for the therapeutic approach and healing process.
PMCID: PMC1774207  PMID: 15306592
Crohn’s disease; fistulae; histology; immunohistochemistry; electron microscopy
3.  Fluorescence endoscopy for the detection of low and high grade dysplasia in ulcerative colitis using systemic or local 5-aminolaevulinic acid sensitisation 
Gut  2003;52(7):1003-1007.
Background and aims: Longstanding ulcerative colitis (UC), especially in the presence of epithelial dysplasia, is associated with an increased risk of developing cancer. As dysplasia is not visible during routine endoscopy, at least 40–50 random biopsies in four quadrants every 10 cm are recommended. Fluorescence endoscopy after sensitisation with 5-aminolaevulinic acid (5-ALA) was assessed for the detection of dysplasia in ulcerative colitis by taking optical guided biopsies. 5-ALA is converted intracellularly into the sensitiser protoporphyrin IX which accumulates selectively in neoplastic tissue allowing the detection of dysplasia by typical red fluorescence after illumination with blue light.
Methods: In 37 patients with UC, 54 examinations were performed with fluorescence endoscopy after oral (20 mg/kg) or local (either with an enema or by spraying the mucosa with a catheter) sensitisation with 5-ALA. A total of 481 biopsies of red fluorescent (n=218) and non-fluorescent (n=263) areas of the colonic mucosa were taken.
Results: Forty two biopsies in 12 patients revealed either low grade (n=40) or high grade (n=2) dysplasia. Sensitivity of fluorescence for dysplastic lesions was excellent and ranged from 87% (95% confidence interval (CI) 0.73–1.00) to 100% (95% CI 1.00–1.00) after local sensitisation, in contrast with only 43% (95% CI 0.17–0.69) after systemic administration. Specificity did not differ for both forms of local sensitisation (enema 51% (95% CI 0.44–0.57) and spray catheter 62% (95% CI 0.51–0.73)); after systemic sensitisation specificity was 73% (95% CI 0.69–0.83). Negative predictive values of non-fluorescent mucosa for exclusion of dysplasia were very high; 89% after systemic sensitisation and 98–100% after local sensitisation. Positive predictive values were 13% and 14% after local sensitisation with enema and spray catheter, and 21% after oral sensitisation with 20 mg/kg ALA. The overall number of biopsies per examination was less than five from fluorescent positive areas.
Conclusion: Fluorescence endoscopy after 5-ALA sensitisation is a possible tool to visualise dysplastic lesions in ulcerative colitis using 5-ALA sensitisation. Local sensitisation is a promising alternative approach compared with systemic administration of 5-ALA. A randomised controlled study is now indicated to compare the efficacy of endoscopic fluorescence detection with the standard technique of four quadrant random biopsies.
PMCID: PMC1773731  PMID: 12801958
ulcerative colitis; dysplasia; fluorescence endoscopy; 5-aminolaevulinic acid
4.  Alterations in p53 predict response to preoperative high dose chemotherapy in patients with gastric cancer 
Molecular Pathology  2003;56(5):286-292.
Aims: To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer.
Methods: In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5–9 of the p53 gene were sequenced directly on ABI 373.
Results: Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients’s sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population.
Conclusion: Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.
PMCID: PMC1187340  PMID: 14514923
gastric cancer; preoperative high dose chemotherapy; molecular parameters; histological regression; p53
5.  Protoporphyrin IX distribution following local application of 5-aminolevulinic acid and its esterified derivatives in the tissue layers of the normal rat colon 
British Journal of Cancer  2001;85(10):1572-1576.
Photodynamic diagnosis and especially therapy after sensitization with 5-aminolevulinic acid (ALA) is hampered by limitations of uptake and distribution of ALA due to its hydrophilic nature. Chemical modification of ALA into its more lipophilic esters seems to be promising to overcome these problems. The aim of the present study was to investigate the comparative kinetics of protoporphyrin IX (PPIX) fluorescence in rat colonic tissue after topical application of ALA and its esterified derivatives, ALA-hexylester (h-ALA), ALA-methylester (m-ALA) and ALA-benzylester (b-ALA). Fluorescence intensity induced by PPIX in normal colonic tissue was quantified using fluorescence microscopy at 1, 2, 4, 6 and 8 h after sensitization. Mucosa exhibited higher fluorescence levels compared to the underlying submucosa or smooth muscle. Peak fluorescence intensities were seen 4 h after local sensitization with 86.0 mol ml−1 ALA (513 ± 0.57 counts per pixel), 6.6 mol ml−1 m-ALA (508 ± 35.50) and 4.8 mol ml−1 h-ALA (532 ± 128.80), and 6 h after sensitization with 4.6 mol ml−1 b-ALA (468 ± 190.27). A 13–18 times lower concentration of ALA esters was required for fluorescence intensities reached with ALA alone. A similar degree of the fluorescence ratio between mucosa and muscularis (5–6:1) was detected for ALA and its derivatives. The time point of the maximum value of this ratio was consistent with peak fluorescence levels for ALA and each ALA-ester. The clinical feasibility and the advantages of topical ALA ester-based fluorescence for detection of malignant and premalignant lesions need further investigations. © 2001 Cancer Research Campaign
PMCID: PMC2363929  PMID: 11720447
5-aminolevulinic acid; 5-aminolevulinic esters; protoporphyrin IX; fluorescence; colon

Results 1-5 (5)