p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus

BACKGROUND & AIMS: The potential role of p16 inactivation by CDKN2A/p16 promoter hypermethylation and/or loss of heterozygosity (LOH) of the CDKN2A gene was investigated in neoplastic progression of Barrett's esophagus. METHODS: CDKN2A promoter hypermethylation was studied by methylation sensitive single-strand conformation analysis and sequencing using bisulfite modified DNA in Barrett's esophageal adenocarcinomas, premalignant lesions, and normal squamous esophageal epithelium. All of the lesions of interest were sampled by microdissection from paraffin-embedded fixed tissue sections. RESULTS: No methylation of the CDKN2A promoter was found in normal esophageal squamous cell epithelia, whereas methylation was detected in 18 of 22 (82%) adenocarcinomas and 10 of 33 (30%) premalignant lesions, including 4 of 12 (33%) samples with intestinal metaplasia only. LOH at the CDKN2A gene locus was found in 68% of adenocarcinomas and in 55% of premalignant lesions. Of 28 samples without p16 immunoreactivity, 25 (89%) showed CDKN2A promoter hypermethylation with or without LOH of CDKN2A. Only 2 (8%) samples expressing p16 protein were found to be methylated; these showed a mixture of completely methylated and unmethylated CDKN2A promoters. In 7 of 19 (37%) informative samples without LOH of CDKN2A, the CDKN2A promoter was found to be methylated at both alleles. Loss of p16 protein expression was strongly associated with CDKN2A promoter hypermethylation (P < 0.00001), but not with LOH (P = 0.33). CONCLUSIONS: Our results indicate that methylation of the CDKN2A promoter is the predominant mechanism for p16 inactivation. This hypermethylation is a very common event in esophageal adenocarcinoma and occurs as early as metaplasia.     

Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma

Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low‐grade and high‐grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high‐grade intraepithelial neoplasia and invasive ESCC, partially occurred in low‐grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere‐specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low‐grade intraepithelial neoplasias, 1.39 ± 0.26 in high‐grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.     

Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia

Mutations in the receptor tyrosine kinase (RTK/RAS) signalling pathway frequently provide a proliferative signal in myeloid malignancies. However, the role of RASSF1A, SHP-1 and SOCS-1, negative regulators of RTK/RAS signalling, has not been extensively investigated in the myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). This study employed methylation-specific polymerase chain reaction (MS-PCR) to determine if aberrant promotor methylation of RASSF1A, SHP-1 and SOCS-1 is involved in the pathogenesis of myeloid malignancies. Patients with MDS (n = 107), AML (n = 154) and juvenile myelomonocytic leukaemia (JMML, n = 5) were investigated, together with 15 normal controls. Primers were located in the promotor region of each gene as well as within exon 2 of SOCS-1. Methylation of RASSF1A was found in five of 55 (9%) MDS cases, but not in any of 57 AML cases studied. RASSF1A methylation was present in one case (20%) of JMML. SHP-1 methylation was present in 13 of 121 (11%) AML cases but was not found in MDS or JMML. SOCS-1 promoter methylation was present in eight of 74 (11%) MDS patients but was not seen in JMML or AML. Importantly, RAS mutations and RASSF1A and SOCS-1 methylation were mutually exclusive indicating that approximately 30% of MDS cases had a defect of the RTK/RAS pathway and its negative regulation. Finally, SOCS-1 exon 2 methylation may not be pathogenetically relevant, since it was detected in samples from normal individuals and did not correlate with promotor methylation.     

The SOCS-1 gene methylation in chronic myeloid leukemia patients

SOCS-1, an important protein in the JAK/STAT pathway, has a role in the down stream of BCR-ABL protein kinase. We investigated 56 CML patients and 16 controls for the methylation status of SOCS-1 gene promoter and Exon 2 regions. Exon 2 was found to be methylated in 58.9% of the patients and 93.8% of the controls [P = 0.020, OR = 0.121(0.015-0.957)%95CI]. The promoter region was found unmethylated in all patient samples and controls. Although previous studies revealed a relation between SOCS1 gene Exon-2 hypermethylation and CML development or progression, the results of this study showed no such correlation. On the contrary, our results might be indicating hypomethylation in CML patients, this hypothesis need to be studied in larger study population.     

Stepwise radical endoscopic resection is effective for complete removal of Barrett's esophagus with early neoplasia: a prospective study

OBJECTIVES: Endoscopic therapy for early neoplasia in Barrett's esophagus (BE) is evolving rapidly. Aim of this study was to prospectively evaluate safety and efficacy of stepwise radical endoscopic resection (ER) of BE containing early neoplasia. METHODS: Patients with early neoplasia (i.e., high-grade intraepithelial neoplasia or early cancer) in BE < or = 5 cm, without signs of submucosal infiltration or lymph node/distant metastases, were included. Patients underwent resection sessions (cap technique after submucosal lifting) with intervals of 6 wk. RESULTS: Between January 2003 and December 2004, 39 consecutive patients were included. Therapy was discontinued in two patients due to unrelated comorbidity. Complete eradication of early neoplasia was achieved in all 37 treated patients in a median number of three sessions. Complete removal of all Barrett's mucosa was achieved in 33 (89%) patients: 4 patients (all had undergone APC [argon plasma coagulation]) were found to have small isles of Barrett's mucosa underneath neosquamous mucosa. Complications occurred in two out of 88 (2%) ER procedures: one asymptomatic perforation, one delayed bleeding. Symptomatic stenosis occurred in 10 of 39 (26%) patients and was effectively treated by endoscopic bougienage. During a median follow-up of 11 months, no patients died and none had recurrence of neoplasia or Barrett's mucosa. CONCLUSIONS: Stepwise radical ER is effective for selected patients with early neoplasia in BE; provides optimal histopathological diagnosis; and may reduce recurrence rate, since all mucosa at risk is effectively removed. Use of APC should be limited to prevent buried Barrett's mucosa. Methods for prevention of stenosis should be developed.     

Barrett's esophagus in scleroderma: increased prevalence and radiographic findings

Ten of 27 patients (37%) with scleroderma who underwent endoscopy at our hospital between 1980 and 1984 for symptoms of reflux esophagitis had biopsy-proven Barrett's esophagus. Two of those 10 patients had esophageal adenocarcinomas. In a blinded review of esophagrams (all but 2 using double-contrast technique) from 16 of the 27 patients, only 1 patient was thought to be at high risk for Barrett's esophagus due to a high esophageal stricture with an adjacent reticular pattern of the mucosa. The latter patient had biopsy-proven Barrett's mucosa. Eight patients were thought to be at moderate risk for Barrett's esophagus due to reflux esophagitis and/or distal strictures in 6 and polypoid intraluminal masses in 2. Three of the 6 patients with esophagitis and/or strictures had Barrett's esophagus, and both patients with masses had adenocarcinomas arising in Barrett's mucosa. Finally, 7 patients who had no esophagitis or strictures were thought to be at low risk for Barrett's esophagus. None of those 7 had histologic evidence of Barrett's mucosa. Thus, the major value of double-contrast esophagography is its ability to classify patients into high-, moderate-, and low-risk for Barrett's esophagus to determine the relative need for endoscopy and biopsy in these patients.     

Aberrant DNA methylation in ulcerative colitis without neoplasia

BACKGROUND/AIMS: DNA methylation has been reported to correlate with the development of colitis associated cancer. We detected the promoter methylation of estrogen receptor gene (ER), TP53, p14, p16, p21 and hMLH1 in ulcerative colitis without neoplasia. METHODOLOGY: A total of 49 specimens from 36 patients, including 36 at rectal inflammatory mucosa and 13 at terminal ileum were obtained by colonoscopic biopsies. Methylation specific polymerase chain reaction were performed to detect the methylation in promoters of the above six genes. RESULTS: Methylation rate of ER promoter was significantly higher in the rectal mucosa than that in the ileum (76.3% vs. 46.2%, P=0.044). Moreover, ER methylation in rectal mucosa was significantly higher in relapse-remitting type compared to one attack only type cases (P=0.008), and also increased in cases longer than 7 years (P=0.036). Methylation rates of p14 or p16 were higher in rectal mucosa than those in the ileum, but the differences were not of statistic significance. Meanwhile, methylation in TP53 promoter was found in only one case, while p21 and hMLH1 methylation were negative in all cases. CONCLUSIONS: Methylation in promoters of ER, p14 and p16 occurs in rectal inflammatory mucosa without neoplasia. Examination of ER methylation in rectal mucosa may be useful for predicting cases at high risk of neoplasia.     

Proliferating cell nuclear antigen in oesophageal diseases; correlation with transforming growth factor alpha expression.

This study was designed to correlate mucosal proliferation in Barrett's oesophagus with expression of a growth promoting peptide, transforming growth factor alpha (TGF alpha). Oesophageal mucosa was studied from 50 patients with oesophageal disease who had been treated by oesophagectomy. Histological analysis showed a range of oesophageal pathology - 18 patients had gastric type Barrett's mucosa, 18 had intestinal type Barrett's mucosa, and 14 had oesophageal adenocarcinomas. Sections were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) (an index of cellular proliferation) and TGF alpha. PCNA immunostaining was seen mainly in the basal cells of the neck/foveolar epithelial compartment of the glands in Barrett's oesophagus. However, in mucosa with high grade dysplasia, the proliferative compartment extended upwards into the superficial layers of the glands. At least 2000 cells were counted in each patient to determine the proportion with PCNA immunoreactivity (PCNA labelling index). The labelling index was highest in adenocarcinoma (25%) and in Barrett's intestinal type mucosa with high grade dysplasia (26%) compared with intestinal type mucosa with no significant dysplasia (20%) and Barrett's gastric type mucosa (12%). There was a significant positive correlation between PCNA labelling indices and TGF alpha expression in Barrett's mucosa (p less than 0.01). In glands showing high grade dysplasia, TGF alpha immunoreactivity was seen in the same regions of the glands as PCNA immunoreactivity, indicating the possibility of involvement of TGF alpha in (pre) neoplastic proliferation in Barrett's oesophagus.     

Relationship between K-ras mutation and the expression of p21WAF1/CIP1 and p53 in chronic pancreatitis and pancreatic adenocarcinoma

Overexpression of p21WAF1/CIP1 was recently described as an early event in the development of pancreatic intraepithelial neoplasia. Since activating K-ras mutations are described in more than 80% of pancreatic cancers and are known to increase intracellular levels of p21WAF1/CIP1 in experimental models, the possible role of activating K-ras mutations in an induction of the p21WAF1/CIP1 expression was investigated in our study. We examined 71 surgical specimens, 29 of chronic pancreatitis and 42 of invasive ductal adenocarcinoma both having a large spectrum of PanIN (pancreatic intraepithelial neoplasia) lesions. Expression of p53 and p21WAF1/CIP1 was examined immunohistochemically and codon 12 K-ras mutational analysis was performed using the very sensitive mutant-enriched PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. Our study demonstrated the overexpression of p21WAF1/CIP1 as an early event in the development of pancreatic intraepithelial neoplasia in the group of chronic pancreatitis and invasive adenocarcinoma as well. Overexpression of p21WAF1/CIP1 increased progressively from normal ducts through the spectrum of PanIN lesions to invasive carcinomas. The p53 overexpression increased again progressively according to the severity of the lesion and seems to be a later event in the development of pancreatic intraepithelial neoplasia if compared to p21WAF1/CIP1 expression. Our results confirmed also the possible p53 independent p21WAF1/CIP1 expression in some PanIN2, PanIN3 lesions and invasive carcinomas. K-ras mutations were not revealed in samples with only low grade PanIN lesions (PanIN1a and PanIN1b). K-ras mutations were detected in 69,4% adenocarcinomas and in only one case of chronic pancreatitis. Two codon 12 K-ras positive pancreatic carcinomas showed K-ras mutations in the surrounding normal pancreatic tissue. In adenocarcinomas, no statistically significant correlation was found between K-ras mutational status and p21WAF1/CIP1 and p53 expression, respectively. The possible role of activating K-ras mutations in an induction of p21WAF1/CIP1 expression was not confirmed in this study.     

Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor

INTRODUCTION Secreted frizzled-related proteins (sFRPs) comprise a family of five secreted glycoproteins that antagonize Wnt canonical and noncanonical signaling by different mechanisms directly or indirectly[1]. Wnt signaling regulates cell growth, motil…     

Aberrant methylation of <Emphasis Type="Italic">SOCS-1</Emphasis> was observed in younger colorectal cancer patients

Background Recently, it was demonstrated that suppressor of cytokine signaling-1 (SOCS-1) was frequently silenced by methylation of its CpG island in human hepatocellular carcinoma (HCC). To define the role of SOCS-1 in the tumorigenic pathway of the colorectum, we examined the methylation of SOCS-1 in tumors of colorectal cancer patients.Methods We examined 74 colorectal cancer patients, using a methylation-specific polymerase chain reaction (PCR; MSP) for SOCS-1 CpG island in primary tumors.Results Aberrant methylation of the SOCS-1 CpG island was detected in 6 of the 74 (8%) colorectal cancer specimens. No corresponding normal colorectal tissues showed SOCS-1 methylation. We then analyzed the correlation between the clinicopathological features and SOCS-1 aberrant methylation and found that younger age was significantly related to SOCS-1 methylation (P = 0.048).Conclusions These findings suggested that SOCS-1 may act as a tumor suppressor in at least some colorectal cancers and that SOCS-1 methylation may be a particular phenomenon related to an early onset of colorectal cancer.     

Activation of c-K-ras mutations in human gastrointestinal tumors

BACKGROUND & AIMS: Ras genes are the most frequently detected oncogenes in human malignancies. Data regarding the frequency of c-K-ras mutations in esophageal, gastric, and small bowel tumors are limited and controversial. METHODS: DNA was extracted from 262 formalin-fixed, paraffin-embedded sections of gastrointestinal samples and tumors, including Barrett's esophagus, esophageal squamous cell carcinomas and adenocarcinomas, and small and large bowel adenomas and adenocarcinomas. The presence of c-K-ras codon 12 mutations was determined using a nonradioactive polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS: c-K-ras mutations were detected in 1 of 39 (2%) patients with Barrett's esophagus, 1 of 21 (5%) adenocarcinomas, 0 of 27 squamous cell carcinomas of the esophagus, and 1 of 32 (3%) gastric adenocarcinomas. It was also present in 8 of 20 (40%) and 10 of 28 (36%) small bowel adenomas and adenocarcinomas, respectively. Similar numbers were observed in 10 of 25 (40%) large bowel adenomas and 11 of 30 adenocarcinomas (37%). Mutations were not associated with age, gender, histology, grade, stage, location, or mortality. CONCLUSIONS: The frequency of codon 12 c-K-ras mutations in small and large bowel tumors is approximately 10-fold higher than that of tumors in the upper gastrointestinal tract.     

p53 overexpression in flat serrated adenomas and flat tubular adenomas of the colorectal mucosa

The expression of the p53 protein was investigated in flat serrated neoplasias as well as in other histological phenotypes of flat or exophytic hyperplasias or neoplasias of the colorectal, mucosa. A total of 104 such lesions were analyzed: 24 were flat serrated neoplasias (22 flat serrated adenomas and 2 flat serrated adenocarcinomas), 26 flat tubular adenomas, 17 flat hyperplastic polyps, 29 exophytic tubular and/or villous neoplasias (23 adenomas and 6 exophytic adenocarcinomas) and the remaining 8, exophytic hyperplastic polyps. Deparaffinized, rehydrated sections were treated immunohistochemically to detect those overexpressing the p53 protein. Lesions having slight (+), moderate (++) or intense (+++) staining were considered immunoreactive. The results showed that 50% of the flat serrated adenomas with low-grade dysplasia (LGD) and 66.7% of those with high-grade dysplasia (HGD) had p53 immunoreactivity. None of the flat tubular or of the exophytic adenomas with LGD expressed p53, but immunoreactivity was present in 61.5% of the flat tubular adenomas with HGD and in 52.3% of the exophytic adenomas with HGD. All adenocarcinomas had an intense p53 reaction. Weak p53 expression was demonstrated by 11.7% of the flat hyperplastic polyps but none of the exophytic polyps reacted. The occurrence of p53 expression in flat serrated adenomas with LGD suggested that, despite its low histological profile, one-half of those lesions could be biologically already committed to independent growth. The occurrence of p53 expression in nearly 12% of the flat hyperplastic polyps was totally unexpected and deserves further investigation. Flat serrated adenoma emerges as a novel, independent histological entity among the various phenotypes of flat neoplasias of the colorectal mucosa.