Endogastric capsule for E-cadherin gene (CDH1) promoter hypermethylation assessment in DNA from gastric juice of diffuse gastric cancer patients.

BACKGROUND: We investigated whether an endogastric capsule (EC) may be a valuable tool for collecting DNA from exfoliated cells from the gastric mucosa and for carrying out an analysis of promoter methylation status of the E-cadherin (CDH1) gene in poorly differentiated, diffuse gastric cancer (DGC). MATERIAL AND METHODS: Consecutive patients with a confirmed diagnosis of poorly differentiated DGC underwent collection of gastric juice by EC. Subjects without cancer and premalignant lesions were also accrued as controls. The samples of gastric juice were processed for DNA isolation and amplification. Then they were used for analysis of CDH1 promoter hypermethylation. RESULTS: The procedure successfully allowed the analysis of CDH1 promoter hypermethylation in 20 patients and 14 controls. This pilot study showed feasibility of the procedure and a significantly different CDH1 promoter hypermethylation status between DGC patients and controls was detected. CONCLUSIONS: The EC may represent an innovative and noninvasive tool for the analysis of a specific epigenetic change in DGC patients. Our findings deserve additional studies as this method may represent a cost-effective tool for early detection of sporadic as well as hereditary DGC in CDH1 germline mutations carriers.

Combined analysis of E-cadherin gene (CDH1) promoter hypermethylation and E-cadherin protein expression in patients with gastric cancer: implications for treatment with demethylating drugs.

BACKGROUND: Hypermethylation is studied as a new, relevant mechanism for silencing tumor suppressor genes. It is a potentially reversible epigenetic change and it is the target of novel anticancer compounds with demethylating activity. In this perspective, we investigated E-cadherin gene (CDH1) promoter hypermethylation in gastric carcinomas and its correlation with E-cadherin protein expression. METHODS: Consecutive cases of gastric carcinoma with assessable paraffin-embedded tumor blocks and paired normal mucosa were considered eligible for study entry. CDH1 promoter hypermethylation and E-cadherin protein expression were determined by methylation-specific polymerase chain reaction and immunohistochemistry, respectively. RESULTS: CDH1 promoter hypermethylation was found in 20 out of 70 gastric carcinomas and the epigenetic change occurred in the early, as well as in the locally advanced disease. In five cases, hypermethylation was also detected in the normal mucosa. Eighteen out of 20 hypermethylated tumors were of the diffuse histotype (P=0.0001). Of 24 tumors with reduced or negative E-cadherin expression, 19 were hypermethylated and 5 were unmethylated (P=0.0001). CONCLUSIONS: CDH1 promoter hypermethylation frequently occurs in gastric carcinomas of the diffuse histotype and it is significantly associated with downregulated E-cadherin expression. The knowledge on the hypermethylation status of tumor suppressor genes may be relevant to the development of demethylating drugs and novel chemopreventive strategies in solid tumors.

An endogastric capsule for measuring tumor markers in gastric juice: an evaluation of the safety and efficacy of a new diagnostic tool.

BACKGROUND: In gastric juice, high levels of the carcinoembryonic antigen (CEA) and the carbohydrate antigen 19-9 (CA 19-9) have been found to correlate with precancerous lesions and gastric cancer. So far, sampling of gastric juice has required upper endoscopy. In place of this invasive procedure, we investigated a new tool for the quantitation of tumor markers in gastric juice. MATERIALS AND METHODS: The study population consisted of healthy controls and consecutive subjects with suspected gastric cancer or dyspepsia/epigastric distress. Patients were asked to swallow a small gelatine capsule (14 mm in length and 5 mm in diameter) containing a pierced plastic cover and surrounding a piece of absorbent paper. The capsule was left in the gastric cavity for 60 min to allow saturation of the absorbent paper with gastric juice. A 45-50 cm length of nylon thread connected to the inner capsule was used to remove the device from the gastric cavity. After processing the absorbent paper for radioimmunoassay, CEA and CA 19-9 levels were correlated to the findings of upper endoscopy and biopsies of gastric mucosa or suspected lesions. RESULTS: The endogastric capsule did not cause any side-effects and 62 participants were fully compliant to the procedure. Assessable gastric juice samples were taken from 23 patients with gastric cancer, 15 patients with intestinal metaplasia or dysplasia, 12 patients with gastritis and 12 controls without gastric diseases. In the 12 samples of gastric juice from control patients, mean values of CEA and CA 19-9 were 1.1 +/- 0.9 ng/ml and 16 +/- 7.5 ng/ml, respectively. The mean levels of both markers were found to increase according to the severity of gastric lesions and in patients with cancer, mean CEA and CA 19-9 levels were 513 +/- 627 ng/ml and 545 +/- 510 ng/ml, respectively. Patients with precancerous lesions and cancer showed higher levels of CEA and CA 19-9 than patients with normal findings or gastritis (P <0.001). CONCLUSIONS: The endogastric capsule is a simple, non-invasive tool for the measurement of CEA and CA 19-9 levels in gastric juice. These values may discriminate between normal or minor pathologic changes and precancerous lesions or carcinomas. Further investigations are warranted, since this may represent a new method for gastric cancer screening.

An analysis of p53, BAX and vascular endothelial growth factor expression in node-positive rectal cancer. Relationships with tumour recurrence and event-free survival of patients treated with adjuvant chemoradiation.

Tumours of patients with node-positive rectal cancer were studied by immunohistochemistry for p53, BAX and vascular endothelial growth factor expressions. Results were correlated to the relapse rate, the pattern of relapse and the event-free survival after radical surgery and adjuvant chemoradiation. After a median follow-up of 60 months, 39 patients remained disease-free and 40 patients relapsed (18 local relapses and 22 distant metastases). The majority of disease-free patients showed p53 negative and vascular endothelial growth factor negative tumours. Local relapses occurred more frequently in patients with p53 overexpressing tumours (P<0.01), while distant metastases were in patients with vascular endothelial growth factor positive tumours (P<0.003). Patients with p53 negative or vascular endothelial growth factor negative tumours showed better event-free survival than patients with p53 positive or vascular endothelial growth factor positive tumours. BAX analysis did not show any association with patients' outcome and it was unrelated to the p53 status. Adjuvant treatment strategies for node-positive rectal cancer may be improved by identifying categories of high-risk patients. In this study, vascular endothelial growth factor and p53 expressions correlated with recurrent disease, pattern of relapse and poor event-free survival.

Potential role and chronology of abnormal expression of the Deleted in Colon Cancer (DCC) and the p53 proteins in the development of gastric cancer.

BACKGROUND: Loss of activity of tumor suppressor genes is considered a fundamental step in a genetic model of carcinogenesis. Altered expression of the p53 and the Deleted in Colon Cancer (DCC) proteins has been described in gastric cancer and this event may have a role in the development of the disease. According to this hypothesis, we investigated the p53 and the DCC proteins expression in different stages of gastric carcinomas. METHODS: An immunohistochemical analysis for detection of p53 and DCC proteins expression was performed in tumor tissue samples of patients with UICC stage I and II gastric cancer. For the purpose of the analysis, the staining results were related to the pathologic data and compared between stage categories. RESULTS: Ninety-four cases of gastric cancer were analyzed. Disease stage categories were pT1N0 in 23 cases, pT2N0 in 20 cases, pT3N0 in 20 cases and pT1-3 with nodal involvement in 31 cases. Stage pT1-2N0 tumors maintained a positive DCC expression while it was abolished in pT3N0 tumors (p <.001). A significant higher proportion of patients with N2 nodal involvement showed DCC negative tumors. In muscular-invading tumors (pT2-3N0) the majority of cases showed p53 overexpression, whereas a significantly higher proportion of cases confined into the mucosa (pT1N0) showed p53 negative tumors. Also, a higher frequency of p53 overexpression was detected in cases with N1 and N2 metastatic lymph nodal involvement. CONCLUSIONS: Altered expression of both DCC and p53 proteins is detectable in gastric carcinomas. It seems that loss of wild-type p53 gene function and consequent p53 overexpression may be involved in early stages of tumor progression while DCC abnormalities are a late event.

Biovularity and "coalescence of primary follicles" in ovaries with mature teratomas.

Several theories have been postulated regarding the origin of ovarian teratomas, including incomplete twinning, neoplastic proliferation of sequestered totipotent blastomeres or primordial cells, derepression of totipotent genetic information in the nuclei of somatic cells, and parthenogenetic development of germ cells. At present parthenogenetic development of ova is the most widely accepted theory, primarily because of the presence of a 46 XX karyotype in almost all mature teratomas. However, some authors have raised the possibility of fusion of ova in the mechanism of formation of ovarian teratomas. We report the results of a study on ovarian tissue adjacent to 31 teratomas to assess the frequency of biovularity, which could provide evidence favoring the last theory. On the whole we found biovularity in 26 ovaries of young patients (mean age, 27 years) with variable numbers of biovular follicles ranging from 1 in 4 cases to more than 10 in 2 cases; the number of biovular follicles depended on the quantity of ovarian tissue examined as well as on the total number of ova in the tissue. In multiple occasions 2 ova were included within a single follicle; in 24 ovaries the biovularity was correlated with coalescence of primary follicles characterized morphologically by an ovoid or hourglass-like shape that resulted from cohesion of 2 follicles. As control cases, 30 ovaries of patients with an average age of 28 years were examined (12 removed for endometriosis, 8 for serous cystadenoma, 7 for tubal pregnancy, and 3 for acute salpingo-oophoritis). Only 1 ovary with endometriosis contained a single biovular follicle. The results suggest that ovarian teratoma development may result from fusion of ova in ovaries containing biovularity and phenomena of coalescence of primary follicles.

Inhibition of vascular endothelial growth factor by octreotide in colorectal cancer patients.

Vascular endothelial growth factor (VEGF) seems to be essential for angiogenesis and for the growth of colorectal cancer; thus its inhibition can arrest tumor growth and decrease metastatic potential. Octreotide has been shown to inhibit growth of colorectal tumors in vitro and in vivo. Part of the antiproliferative activity of octreotide could be related to its antiangiogenic properties. Effects of octreotide on VEGF expression were evaluated in 35 patients with operable colorectal cancer receiving octreotide for 2 weeks before surgery. Tissue VEGF expression and serum VEGF concentrations were determined before and after treatment with octreotide. There was a statistically significant reduction in the tissue VEGF expression both considering the percentage of VEGF positive cells (P = 0.006) and the intensity of VEGF staining (P = 0.003). A similar significant reduction was observed in serum values of VEGF (P = 0.03). The present study indicates that octreotide inhibits expression of VEGF in colorectal cancer patients, and, furthermore, that serum VEGF expression correlates with tissue VEGF, representing a safe method to monitor the activity of antiangiogenic agents.

Vascular endothelial growth factor expression, S-phase fraction and thymidylate synthase quantitation in node-positive colon cancer: relationships with tumor recurrence and resistance to adjuvant chemotherapy.

BACKGROUND: The behaviour of colorectal carcinomas may depend on molecular properties of tumors. In node-positive colon cancer, we assessed the S-phase fraction (SPF) index, the vascular endothelial growth factor (VEGF) expression and the TS levels. The combined analysis of SPF/VEGF was studied for predictivity of recurrent disease, the TS quantitation was related to the efficacy of fluorouracil-based adjuvant chemotherapy. PATIENTS AND METHODS: Consecutive patients with surgically-resected, node-positive colon cancer were studied. Flow cytometry for the SPF and immunohistochemistries for the TS and the VEGF expression were carried out on the primary tumor. Recurrences had to be proven by biopsy or surgery, and they were categorized as early, if occurred within 12 months after surgery, or late if occured 13 months or more. RESULTS: Of 150 evaluable patients, 100 had received fluorouracil-based adjuvant chemotherapy and 50 control patients were untreated. The combined analysis of the VEGF and the SPF showed a strong association between the two markers; 48 patients (32%) had high SPF/VEGF positive tumors and 69 patients (46%) had low SPF/VEGF negative tumors (P < 0.0001). The majority of disease-free patients (73.4%) showed VEGF negative/low SPF tumors (P < 0.0001). Early recurrences occurred more frequently in patients with VEGF positive/high SPF tumors (P < 0.001). In the 100 patients treated with adjuvant chemotherapy, 86% of relapsed patients had TS overexpressing tumors and 69% of disease-free patients had TS negative tumors (P < 0.001). Also, early recurrences occurred more frequently in TS overexpressing tumors (P < 0.0001). CONCLUSIONS: Evidence is supported that node-positive colon cancer constitutes a heterogenous disease. Patients with VEGF positive/high SPF tumors showed an unfavourable outcome compared to patients with VEGF negative/low SPF tumors. The efficacy of fluorouracil-based adjuvant chemotherapy may depend on the TS status.

Immunohistochemical determination of p53 protein does not predict clinical response in advanced colorectal cancer with low thymidylate synthase expression receiving a bolus 5-fluorouracil-leucovorin combination.

BACKGROUND: We assessed the hypothesis that a compromised p53 function could account for the non response of colon cancer patients with low thymidylate synthase (TS) expression receiving a bolus 5-fluorouracil (5-FU) leucovorin (LV) combination. PATIENTS AND METHODS: The study population consisted of 41 patients with unresectable metastatic colon cancer, homogeneously, treated with bolus 5-FU and LV. RESULTS: Twenty-seven patients (66%) showed high levels of TS expression. The difference in the proportion of objective responses between patients with low (CR + PR: 7 of 14, 50%) and high (CR + PR: 0 of 27) TS levels was statistically significant (P = 0.0001, chi-square test). p53 nuclear over-expression was found in 27 of 41 patients (66%). No differences were observed in p53 overexpression in patients with high (66%) or low (66%) TS expression. p53 status was not found to be associated with response even in patients with low TS expression. CONCLUSIONS: p53 status measured by immunohistochemistry does not seem to be useful to identify unresponsive patients with low TS expression.

Expression of vascular endothelial growth factor can predict event-free survival in stage II colon cancer.

The usefulness of chemotherapy in patients with stage II disease continues to be debated. Biological prognostic factors may allow further insight into the optimal treatment strategy for patients with node-negative disease. Vascular endothelial growth factor (VEGF) seems to be essential for angiogenesis and for the growth of colorectal cancer. Recently, it was shown able to predict disease recurrence in patients with stage II colon cancer. Specimens of surgically resected colon cancer were immunostained for VEGF. Consecutive patients referred to the study institutions were considered eligible for this study. The main inclusion criteria were stage II tumor, sufficient tumor material, and adequate follow-up information. Analysis was performed on 121 patients. The recurrence rate in the patients with VEGF-positive tumors was 50% (18 of 36 patients), which was significantly higher than that observed in patients with VEGF-negative tumors [11.7% (10 of 85 patients); P = 0.001]. Also the degree of VEGF immunoreactivity was significantly higher in 28 relapsing patients compared with 93 disease-free patients (mean VEGF score, 2.84 0.38 versus 0.66 +/- 0.17; P = 0.0001). VEGF may be used in a clinical setting to identify patients at high risk for relapse who may benefit from adjuvant treatment including new therapeutic strategies such as monoclonal antibody neutralizing VEGF.

Effects of calcium and vitamin supplementation on colon cell proliferation in colorectal cancer.

Calcium and antioxidant vitamins, such as A, C, and E, have been shown to reduce colorectal epithelial proliferation and thereby to act as possible chemoprotective agents in colorectal cancer. We investigated the effects of an intervention with calcium and vitamins on cell proliferation in the colonic mucosa of patients operated on for colorectal cancer. Patients with resected colorectal cancer Dukes' stage B-C were randomized to receive daily 30,000 IU of axerophthol palmitate (vitamin A) plus 1 g ascorbic acid (vitamin C) plus 70 mg of dl-alpha-tocopherol acetate (vitamin E) and 2 g natural calcium daily or indistinguishable placebo for 6 months. At the time of surgery and after 6 and 12 months of treatment, cell kinetics of normal colonic mucosa were assessed by using proliferating cell nuclear antigen (PCNA). Ninety patients were enrolled and 77 were assessable: 34 in the treatment group and 43 in the placebo group. A significant reduction of mean total PCNA labeling index (PCNALI) was evident in both groups after 6 months (vitamins/calcium, from 16.11 +/- 2.43 to 10.71 +/- 2.81; placebo, from 17.30 +/- 2.63 to 12.53 +/- 3.40). The difference in the percentage of reduction of mean PCNALI between baseline and after 6 months was not statistically significant in the treatment and placebo groups: 34% and 28%, respectively. A second control, 6 months after discontinuation of vitamin and calcium supplementation, showed a further decrease of mean total PCNALI in both groups, but this was not statistically significant. Our randomized trial showed that calcium and vitamin supplementation does not reduce cell kinetics of colon epithelium. Furthermore, this study suggests the need for extreme caution in the interpretation and publication of studies on chemoprotectants in colon cancer without a control group.

Unusual bone marrow manifestations of parvovirus B19 infection in immunocompromised patients.

Parvovirus B19 is responsible for a spectrum of disease in humans. The usual bone marrow findings in acute parvovirus infections are marked erythroid hypoplasia and occasional giant erythroblasts. Intranuclear inclusions in developing erythroid precursors are rarely described in children or adults with parvovirus infection, although abundant intranuclear inclusions are commonly observed in the placenta and other tissues in infected fetuses. In this study, 8 patients are reported in whom the first evidence of parvovirus infection was the recognition of numerous intranuclear inclusions in erythroid precursors on bone marrow biopsy sections. Six of the 8 patients had documented immunodeficiencies; 4 had acquired immune deficiency syndrome (AIDS), and 2 were on chemotherapy. Five of 7 patients were negative for immunoglobulin G (IgG) antiparvovirus antibodies, including all 4 with AIDS. Unlike the typical pattern in parvovirus infection, the bone marrow was hypercellular in most of the patients, and erythroid precursors were usually increased with the entire spectrum of normoblast maturation represented; abundant intranuclear inclusions were observed similar to the finding in fetuses. The inclusions were variably eosinophilic and compressed the chromatin against the nuclear membrane. In situ hybridization showed parvovirus B19 DNA in numerous erythroid precursors in all cases. The findings of erythroid maturation and abundant viral inclusions in these immunocompromised patients is consistent with the hypothesis that failure to produce effective IgG parvovirus neutralizing antibodies may lead to persistent infection through viral tolerance that allows erythroid development of infected cells past the pronormoblast stage. Identification of parvovirus inclusions in marrow biopsies and subsequent confirmation of infection by in situ hybridization can be important in the assessment of anemia in immunodeficient patients because serological studies for parvovirus B19 are frequently negative.

Differences of vascular endothelial growth factor (VEGF) expression between liver and abdominal metastases from colon cancer. Implications for the treatment with VEGF inhibitors.

The vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis, and it is the target of innovative anti-cancer therapies. In colorectal carcinomas, differences in the VEGF expression have been found between the primary tumor and its metastases. We postulated that differences in the VEGF expression may also exist between liver and abdominal metastases from colon cancer. Consecutive colon cancer patients with liver or abdominal metastases were considered eligible for the study. Biopsies had to be performed before chemotherapy and the VEGF analysis were conducted through immunohistochemistry. The staining results were correlated to the metastatic pattern. The study population consisted of 41 patients with a metastatic site in the liver in 19 patients and the abdomen in 22 patients. A positive VEGF staining was found in 19 of the 41 metastatic samples (46%). Cases with positive VEGF expression were found more frequently in abdominal (15 out of 22 patients; 68%) than in liver metastases (4 out of 19 patients; 21%). Also, the degree of VEGF immunoreactivity was significantly higher in abdominal than in liver metastases. Evidence is supported that the VEGF expression may be different between colon cancer metastatic sites. The efficacy of anti-VEGF treatments may depend on the VEGF expression status, and this finding deserves further investigation.

Regression of multiple hepatic masses in a young patient with thalassaemia.

BACKGROUND: We report a young girl with thalassaemia who showed development and regression of multiple hepatic masses. RESULTS: The tumours, detected 3 years after allogeneic bone-marrow transplantation, showed progressive reduction in size and number following a phlebotomy program to treat iron overload. CONCLUSION: The detailed CT, MRI and histological findings are described.

Thymidylate synthase protein expression in advanced colon cancer: correlation with the site of metastasis and the clinical response to leucovorin-modulated bolus 5-fluorouracil.

Recently, we have demonstrated that thymidylate synthase (TS) protein expression predicts for the clinical response to a regimen of infusional 5-fluorouracil (5FU) in advanced colorectal cancer patients. Previous studies by other groups that showed a correlation between TS gene expression and response to the fluoropyrimidine also involved infusional regimens. Considering the putatively different mechanism of action of bolus compared with continuous infusion of 5FU, the aim of the present study was to test whether the correlation between TS expression and the clinical response to 5FU is valid for bolus regimens. A secondary aim was to compare TS levels between liver metastases and abdominal recurrences from colon cancer, because these sites have a distinctly different responsiveness to 5FU chemotherapy. The study population consisted of 41 patients (25 males and 16 females; median age, 60 years) with unresectable metastatic or recurrent colon cancer, homogeneously treated with 5FU (420 mg/m2 i.v., days 1-5) and leucovorin (20 mg/m2 i.v., days 1-5); cycles were repeated every 28 days. Twenty-seven patients (66%) showed high levels of TS expression as defined by TS scores equal to 3 and 4. The proportion of cases with high levels of TS expression was significantly higher in abdominal recurrences (18 of 22, 82%) compared with liver metastases (9 of 19, 47%; P = 0.02). Intratumoral TS protein expression was inversely correlated with response to chemotherapy (response rate: 7 of 14, 50%, versus 0 of 27 in patients with low and high levels of TS expression, respectively; P = 0.0001). These results confirm that the level of TS protein expression predicts for response to 5FU, even with a bolus schedule. The higher TS levels observed in abdominal compared with liver metastases may account for their different responsiveness to 5FU chemotherapy. Immunohistochemical quantitation of TS protein levels may thus allow us to change the therapeutic approach to advanced colorectal cancer from a general to an individual treatment strategy at a time when new non TS-targeted drugs have become available for this disease.

Bone marrow transplantation in adult thalassemic patients.

One hundred seven adult patients with thalassemia aged from 17 through 35 years and transplanted from HLA-identical siblings between November 1988 and September 1996 were evaluated on December 31, 1997. The outcome experience of 20 consecutive patients transplanted between November 13, 1988 and January 10, 1991 and reported in September 1992 is updated after 5 additional years. The experience on 87 patients transplanted between May 1991 and September 1996 is described and evaluated as of the end of December 1997. Of 107 patients, 69 survive between 1.5 and 9 years after transplantation. Sixty-six of these patients do not have thalassemia and are identified as ex-thalassemic after bone marrow transplantation. The youngest survivor is 20 years old, 6 are older than 30 years, and the oldest is 37 years of age. Patients with chronic active hepatitis at the time of transplant were significantly more likely to die than patients without (P =.05; relative risk, 2.05). Marrow transplantation is a valid treatment option for older patients with thalassemia who have suitable donors and show deterioration with conventional therapy.