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.

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.

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.

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.

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.

Ultrasound-guided fine needle cutting biopsy for the characterization of diffuse liver diseases in bone marrow transplant patients.

Liver disease is a frequent complication in bone marrow transplant recipients and may occur early or late in the post-transplant period. Using ultrasound-guided fine needle (1.2 mm, 18 G) cutting biopsy, we studied six patients with undefined late post-BMT liver disease. No procedure-related complications occurred and all liver biopsies were informative, leading to changes in therapeutic approach. In our small series, the most frequent cause of hepatic damage was drug toxicity. US-guided fine needle cutting biopsy is a useful and easy tool for the work-up of unexplained post-BMT liver disease.

Kaposi's sarcoma after allogeneic bone marrow transplantation.

A case of Kaposi's sarcoma (KS) in an allogenic BMT recipient is reported. A 26-year-old man underwent allogeneic bone marrow transplantation for microdrepanocytosis. He received prolonged immunosuppressive therapy for mild chronic GVHD. Two years after BMT he developed KS localized to the skin. The KS improved rapidly and outcome was complete remission after cessation of immunosuppression.

Mucormycosis after bone marrow transplantation: report of four cases in thalassemia and review of the literature.

We report four cases of mucormycosis that occurred among 711 patients who underwent BMT for thalassemia, and review 18 additional cases among BMT recipients that were reported in the English-language literature. All these patients were polytransfused and were in advanced phase of disease with severe acquired hemochromatosis. The sites of infection were sinonasal, rhinocerebral-pulmonary, pulmonary and pulmonary-central nervous system. Mucormycosis was the primary cause of death in three of four patients. Two infections were detected within the first 100 days after BMT. Only one of the four patients had partial resolution of sinonasal mucormycosis following aggressive antifungal therapy combined with hyperbaric oxygen treatment.

Alpha-interferon treatment of chronic hepatitis C after bone marrow transplantation for homozygous beta-thalassemia.

No experience has been reported to date in treating chronic hepatitis C virus (HCV) infection with interferon (IFN) therapy after BMT, mainly due to concerns related to the impact of an immunomodulatory drug in patients who are immunologic and haematologic chimeras. However, chronic inflammatory activity related to HCV infection results in a chronic fibrogenous mechanism potentially leading to liver cirrhosis and hepatocellular carcinoma. Moreover, patients transplanted for beta-thalassemia could be at greater risk because of concomitant iron overload and pre-existing fibrous liver damage. Eleven patients with serological, biochemical, histological and molecular biological evidence of HCV infection were included in the study and treated for 6-12 months with recombinant IFN 24-65 months following BMT. The serum alanine aminotransferase (ALT) was persistently elevated (range 85-1242 U/l; mean 416) for at least 1 year prior to IFN treatment. Ten patients completed the protocol; five were considered as responders to treatment. In these five patients the liver histology showed an overall reduction of inflammation and necrosis: histological inflammatory activity improved from chronic active hepatitis (CAH) to chronic persistent hepatitis (three patients) or minimal residual inflammatory activity (two patients). The Knodell total activity score varied from 5.4 (range 3-9) to 1.4 (range 1-2; P = 0.05). All responding patients revealed negativization of serum HCV-RNA, that has been persistent in four (follow-up 1-3 years). ALT level fell to 15-80 U/l (mean 52; P = 0.0027). No major complications occurred during the therapy and no influence on marrow engraftment parameters were noted. We conclude that IFN therapy does not adversely interfere with engraftment and that it is a feasible therapy for treatment of chronic hepatitis C virus after BMT.

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.

Chronic liver disease and active hepatitis C virus infection in patients with antibodies to this virus.

AIMS: To assess the association between active hepatitis C virus (HCV) infection and liver damage in randomly selected patients with antibodies to the virus. METHODS: Thirty three consecutive subjects with serologically confirmed positivity for antibodies to HCV were studied for the presence of liver and circulating viral sequences by using the reverse transcription polymerase chain reaction (RT-PCR) and specific primers for the 5'-untranslated region (5'-UTR) of the HCV genome. Parallel clinical, biochemical, and histological investigations were carried out in all cases. RESULTS: A comparative virological and histological investigation showed the presence of molecular signs of active viral replication and different degrees of liver damage in all cases. Baseline values of liver and plasma samples from all the patients showed (with one exception) the presence of detectable HCV RNA sequences, despite alanine amino transferase activities being within normal values or within 1.5 times the upper limit of normal in 13 of them. Examination of percutaneous liver biopsy specimens showed the presence of confirmed liver damage (ranging from chronic persistent hepatitis to cirrhosis) in all 33 patients. CONCLUSIONS: Circulating HCV RNA sequences (a direct sign of active HCV infection) are associated with liver damage, even in the absence of clinical or biochemical signs of overt liver disease. Parallel molecular, histological, and clinical follow up of these patients is needed to understand precisely the natural history of HCV infection and for correct clinical management.

Treatment of iron overload in the "ex-thalassemic". Report from the phlebotomy program.

After successful marrow transplantation (BMT) iron overload remains an important cause of morbidity in Thalassemia. After BMT, patients have normal erythropoiesis capable of producing a hyperplastic response to phlebotomy so that this procedure can be contemplated as a method of mobilizing iron from overloaded tissues. Forty-one patients (mean age 16 +/- 2.9 years) with prolonged follow-up (range 2-7 years) after BMT were submitted to a moderate intensity phlebotomy program (6 ml/kg blood withdrawal at 14-day intervals) to reduce iron overload. Values are expressed as mean +/- SD or as median with a range (25th-75th percentile). Serum ferritin decreased from 2,587 (2,129-4,817) to 280 (132-920) micrograms/l (p < 0.0001), total transferrin increased from 2.34 +/- 0.37 to 2.9 +/- 0.66 g/l (p = 0.0001), transferrin saturation decreased from 90% +/- 14% to 39% +/- 34% (p < 0.0001). Liver iron concentration evaluated on liver biopsy specimens decreased from 20.8 (15.5-28.1) to 3 (0.9-14.6) mg/g dry weight (p < 0.0001). Alanine amino-transaminase from 5.2 +/- 3.4 to 1.6 +/- 1.2 (p < 0.0001) times the upper level of normality. The histological grading for chronic hepatitis (Histology Activity Index) decreased from 4.2 +/- 2.4 to 2.3 +/- 1.8 (p < 0.0001). Phlebotomy is a safe, efficient, and widely applicable method to decrease iron overload in "ex-thalassemic."

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.

An immunohistochemical study on foetuses and newborns lymph nodes with emphasis on follicular dendritic reticulum cells.

Lymph nodes were withdrawn from twenty foetuses and eighteen newborns. Paraffin embedded tissues were stained by current histochemical methods and immunohistochemically processed with a panel of antibodies against dendritic reticulum cells (DRC) (CD21, CD35), endothelial cells (QBEnd/10-CD34, CD31, Factor VIII RAg), B lymphoid cells (L26, MB2), T lymphoid cells (UCHL1, DF-T1, OPD4), monocytic cells (PGM1, KP1) and interdigitating dendritic cells (S100 protein, PGM1). The immunohistochemistry was focused on determining the initial appearance of DRC whose origin is still debated. They are currently considered to be transformed mesenchymal cells. This study shows that DRC make their appearance in the superficial or subcutaneous lymph nodes just at the beginning of extrauterine life whereas they are identifiable in the mesenteric lymph-nodes already during the foetal life. They acting as an enhancing microenvironment, appear to be related to B lymphocyte expansion. Their derivation from endothelial cells rather than other mesenchymal cells seems more probable.

Lymphoid follicles in extranodal sites: an immunohistochemical study.

Lymphoid follicles in extranodal sites involved in chronic inflammatory reactions are the subject of this study. Immunohistochemistry was focused on associated lymphoid formations considered as incomplete or prefollicular structures because they were composed only of some types of the germinal centre cells among demonstrated dendritic reticulum cells (DRC) and thin vessels. Paraffin embedded tissues were stained with current histological methods and immunohistochemically processed with a panel of antibodies against DRC (CD21, CD35, IgM); endothelial cells (factor VIII R. Ag., EUA); B lymphoid cells (L26, MB2); T lymphoid cells (UCHL1, MT1): intercellular adhesion molecule (ICAM-1); histiocytic/monocytic cells (KP1, PS100, delta-1-antitrypsin, delta-1-antichymotrypsin); smooth muscle (actin 1A4); basement membranes (collagen IV). Prefollicular structures showed positivity for B cells markers among a network of DRC and thin long processes. Furthermore, scattered T lymphoid and histiocytic cells such as collagen IV fragments and some pericytic cells were demonstrated. Some sections with Emat-eos. were destained and submitted to immunohistochemistry analyses: in prefollicular structures, histologically referred endothelial cells were positive for dendritic reticulum markers and ICAM-1. These findings appear to favor the hypothesis that DRC could be transformed endothelial cells which therefore represent an important component in follicular formation.

Immunohistochemical study of tonsils from newborn infants with emphasis on follicular dendritic reticulum cells.

The origin of follicular dendritic reticulum cells (FDRCs) is still debated in the literature, although their derivation from local transformed mesenchymal cells is now generally accepted. The purpose of this immunohistochemical study was to further define the nature of the FDRC, for which an endothelial cell derivation was proposed in previous papers. Palatine tonsils were removed at autopsy from eight newborn infants ranging from a few hours to four days of age. Paraffin embedded tonsil specimens were stained by current histochemical methods and immunohistochemically processed with a panel of antibodies against follicular dendritic reticulum cells (CD21 and CD35), endothelial cells (Factor VIII R. Ag, CD31, CD34), B lymphoid cells (L26, LN1, MB2), T lymphoid cells (UCHL1, DF-T1, OPD4, CD8, CD3), monocytic cells (PGM1 and KP1), interdigitating dendritic cells (S100 Protein, PGM1), intercellular adhesion molecule (ICAM-1 (CD54). Double immunostainings for endothelial markers and for FDRCs were performed. In newborn infant tonsils, lymphoid follicles are absent. T lymphocytes appear to represent the largest component whereas B lymphocytes are seen in small aggregates between blood capillary vessels. Monocytes and interdigitating dendritic cells are also present. The capillary endothelial cells within the B lymphoid aggregates are positive for endothelial and FDRCs markers; double immunostaining for Factor VIII R Ag (or other endothelial markers) and CD21 was present within the same capillary endothelial cells. Further they were positive for ICAM-1. These observations lend further evidence that the derivation of FDRCs may be from transformed endothelial cells and on this way they act as enhancing microenvironment for B lymphocyte expansion.

Budd-Chiari syndrome complicating lung carcinoma of the right inferior lobe: a case report.

A case of bronchogenic carcinoma involving the base of the right inferior lobe and causing Budd-Chiari syndrome in a 66-year-old woman is reported. It appears to be the second case in the literature with a similar clinical manifestation.

"Angioblastic" adamantinoma of the tibia. An immunohistochemical study.

An "angioblastic" adamantinoma of the tibia is described in a 78-year-old man. The diagnosis was defined by immunohistochemical methods, which showed a positivity for keratin in the cells lining spaces with a vascular appearance.

Hepatocellular carcinoma with intracytoplasmic hyaline globules.

A case of hepatocellular carcinoma with intracytoplasmic hyaline globules is reported. By serological, histochemical, immunofluorescent and electron microscopic studies the authors maintain that in this case intracytoplasmic hyaline globules represent lysosomal structures. According to Von Ardenne's theory, these lysosomal globules appear related to neoplastic cells necrosis.

Giant lymph node hyperplasia in angiohamartomatous soft tissues.

The first case of Castleman's benign lymphoma associated with angiohamartomatous alterations in peripheral fibroadipose tissue is reported, and a pathogenetic connection is hypothesized. The lesion was encountered in a 48-year-old man; it was situated in the left and inferior retroperitoneal area with extension to the inguinal region of the same side.