Haematobium eggs detection in human bladder cancer and sporocysts in snail vectors: Seven cases report and a review of the Burkina Faso literature.

Schistosoma haematobium plays a central role in the development of bladder cancer in Burkina Faso. The objective of this study was to determine the presence of S. haematobium in the bladder cancer and in vector snails. For the first time, formalin-fixed tissues embedded in paraffin were analyzed by immunohistochemistry and PCR. Molecular detection has resulted in 7/7 positive bladder cancer. Finally, as the snail vectors were positive. We suggest the use of molecular methods in the snail vectors for the detection of cysts and in cancerous tissues in larger studies.

Application of alternative fixatives to formalin in diagnostic pathology.

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose.

Exposure during early development to chemicals with hormonal action may be associated with weight gain during adulthood because of altered body homeostasis. It is known that organotins affect adipose mass when exposure occurs during fetal development, although no knowledge of effects are available for exposures after birth. Here we show that the environmental organotin tributyltin chloride (TBT) exerts adipogenic action when peripubertal and sexually mature mice are exposed to the chemical. The duration and extent of these effects depend on the sex and on the dose of the compound, and the effects are relevant at doses close to the estimated human intake (0.5µg/kg). At higher doses (50-500µg/kg), TBT also activated estrogen receptors (ERs) in adipose cells in vitro and in vivo, based on results from acute and longitudinal studies in ERE/luciferase reporter mice. In 3T3-L1 cells (which have no ERs), transiently transfected with the ERE-dependent reporter plus or minus ERα or ERß, TBT (in a dose range of 1-100nM) directly targets each ER subtype in a receptor-specific manner through a direct mechanism mediated by ERα in undifferentiated preadipocytic cells and by ERß in differentiating adipocytes. The ER antagonist ICI-182,780 inhibits this effect. In summary, the results of this work suggest that TBT is adipogenic at all ages and in both sexes and that it might be an ER activator in fat cells. These findings might help to resolve the apparent paradox of an adipogenic chemical being also an estrogen receptor activator by showing that the two apparently opposite actions are separated by the different doses to which the organism is exposed.

Chromosome 9 instability and alterations of p16 gene in squamous cell carcinoma of the lung and in adjacent normal bronchi: FISH and immunohistochemical study.

AIMS: p16, a tumour suppressor gene located at 9p21 chromosome and involved in cell cycle regulation, is often inactivated in lung carcinoma. Inactivation is also supported by the loss of p16 protein, a strong inhibitor of cyclin-dependent kinase (CDK) 4 and 6. The aim of this study was to examine alterations of p16 both in pulmonary squamous cell carcinoma (SCC) and in morphological normal bronchi contiguous with neoplasia. METHODS AND RESULTS: p16 gene and chromosome 9 alterations were examined by fluorescence in situ hybridization and the expression of p16 protein by immunohistochemistry in pulmonary surgical specimens from 31 patients with SCC. As controls, surgical specimens from 13 patients with non-neoplastic pathology were examined. Tumours showed molecular alterations for p16 gene and chromosome 9 abnormalities in, respectively, 29/31 and 19/31 cases respectively. p16 protein was unexpressed in 29/31 cases. In morphologically normal bronchi p16 gene and chromosome 9 alterations occurred in, respectively, 13/31 and 4/31 cases respectively; loss of protein immunoreactivity occurred in 14/31 cases. No alterations were seen in any of the control cases. CONCLUSIONS: Inactivation of p16 gene in histologically normal bronchi could aid the identification of individuals at risk of developing SCC of the lung.

HER-2/neu overexpression and amplification in uterine serous papillary carcinoma: comparative analysis of immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and fluorescence in situ hybridization.

Uterine serous papillary carcinoma (USPC) is a rare and highly malignant form of endometrial cancer (EC) characterized by early metastasis, chemoresistance, and high mortality rate. Little is known about USPC tumorigenesis even if recently a HER-2/neu role has been suggested in its development and progression. The aim of the present study was to evaluate HER-2 expression by immunohistochemistry (IHC) in 12 USPC formalin-fixed, paraffin-embedded (FFPE) samples. Moreover, we looked at the correlation between HER-2 protein expression and HER-2/neu gene amplification by fluorescence in situ hybridization (FISH), other than HER-2/neu messenger RNA expression by quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR). Finally, these results have been compared with commonly evaluated clinical features in EC patients, in order to define the potential prognostic value of HER-2/neu overexpression in USPCs. A high expression of HER-2 protein by IHC was noted in 2 of 12 patients (16.6%), and the same cases showed specific HER-2/neu gene amplification by FISH. All the samples investigated displayed a perfect concordance between IHC and FISH data. Five (41.6%) of 12 tumors demonstrated polysomy of chromosome 17 and, focusing on the 2 USPCs that showed HER-2/neu overexpression, one of them (50%) was polysomic for chromosome 17. All the other USPC cases (58.4%) showed to be disomic for chromosome 17. Quantitative RT real-time PCR performed on complementary DNA obtained from all FFPE USPC samples showed a complete correlation with FISH and IHC data. Moreover, HER-2/neu overexpression was associated with a poorer overall survival and a very low relapse-free survival time, thus being considered a candidate marker of worse overall prognosis in USPC. The use of trastuzumab (Herceptin), a monoclonal antibody directed against HER-2/neu, for the therapy of patients with HER-2/neu-positive USPCs should be further investigated in clinical trials.

Chromosomal study of enteric glial cells and neurons by fluorescence in situ hybridization in slow transit constipation.

The pathogenesis of slow transit constipation is still elusive. However, a genetic basis may be present. We investigated possible chromosomal abnormalities in enteric neurons and glial cells in patients with slow transit constipation. Colonic specimens from 22 patients with slow transit constipation undergoing surgery for intractable symptoms were obtained, and investigated by fluorescence in situ hybridization (FISH) for chromosomal abnormalities (chromosomes 1, 8, 17 and XY). These specimens were compared with of those obtained in 12 control subjects. Data analysis showed that 45.5% of patients displayed significant (>10%) aneusomy of chromosome 1 in enteric neurons. Aneusomy <10% for the same chromosome, but less than the cutoff suggested (10%), was found in enteric glial cells in 45.4% of the same patients. One patient had <10% aneusomy in enteric neurons for chromosome 8. No other abnormalities were found for the remaining probes, and no abnormalities were found in controls. We concluded that in a subgroup of patients with slow transit constipation a genetic basis may be present.

Genistein affects adipose tissue deposition in a dose-dependent and gender-specific manner.

The soy isoflavone genistein targets adipose tissue and elicits physiological effects that may vary based on dietary intake. We hypothesized that the adipose effects of genistein are dose and gender dependent. Four-week-old C57BL/6 male and female mice received daily oral doses of genistein (50-200,000 microg/kg.d) or 17beta-estradiol (E2) (5 microg/kg.d) for 15 d or a diet containing 800 ppm genistein. Genistein increased epididymal and renal fat pad and adipocyte size at doses up to 50,000 microg/kg.d or at 800 ppm in the diet in males but not in females. The alteration in adipocity correlated with changes in peripheral insulin resistance. These treatments increased genistein serum concentrations from 35+/-6 to 103+/-26 nM 12 h after treatment and lowered plasma triglycerides and cholesterol levels. The 200,000 microg/kg.d genistein dose decreased adipose tissue weight similarly to E2. This genistein dose down-regulated estrogen receptor (beta more than alpha) and progesterone receptor expression and induced estrogen-dependent adipose differentiation factors; it did not change expression of the minimal consensus estrogen-responsive element in ERE-tK-LUC mice, which was positively modulated in other tissues (e.g. the lung). E2 down-regulated almost all examined adipogenic factors. Gene microarray analysis identified factors in fat metabolism and obesity-related phenotypes differentially regulated by low and high doses of genistein, uncovering its adipogenic and antiadipogenic actions. The lower dose induced the phospholipase A2 group 7 and the phospholipid transfer protein genes; the 200,000 microg/kg.d dose inhibited them. The antiadipogenic action of genistein and down-regulation of adipogenic genes required the expression of ERbeta. In conclusion, nutritional doses of genistein are adipogenic in a gender-specific manner, whereas pharmacological doses inhibited adipose deposition.

Spectrophotometric assessment of nuclear proteins: a preliminary study.

Qualitative evaluation of protein content in formalin fixed, paraffin-embedded tissues is usually performed by means of cytofluorimetric analysis. On the other hand, several studies underline the opportunity to measure the concentration of nuclear proteins, which is often accomplished by using complex techniques and instrumentation. In the present work, we suggest a new application for the spectrophotometric evaluation of protein content on extracted and isolated nuclei, based on EDTA treatment of specimens and chemical extraction of proteins, followed by direct spectrophotometric measurement at UV wavelengths. We also demonstrate how this parameter correlates with other diagnostic factors, such as the proliferation index (MIB-1) and the DNA content (ploidy) of cells. This method is simple and effective, yet less expensive than other protein quantitation protocols.

[Limitations of colonic lavage in the cytopathological diagnosis of inflammatory and neoplastic lesions]. / Limitazioni del lavaggio colico nella diagnostica citopatologica di lesioni neoplastiche e infiammatorie.

The authors describe the personal experience on colonic lavage cytology in neoplastic or inflammatory diseases. Although the presence of false negative results in the lesions of right colon, the method could be useful in the diagnosis of selected cases.

The collecting duct carcinoma of the kidney: a cytogenetical study.

OBJECTIVES: The Heidelberg classification of renal tumours identifies five histotypes of renal cancer, underlining for two of them (conventional and papillary renal cancers) a strict relation between the morphological aspect and the complement of alterations evidenced by the cytogenetic analysis of the neoplastic karyotype. Due to its low incidence, the collecting duct carcinoma (CDC) has not yet been characterized from a cytogenetic point of view. This study analyses the clinical, morphologic and cytogenetic features of the CDC observed and treated in our department. METHODS: From January 1995 to December 2002, among the 591 patients who underwent surgery for renal cancer, we observed 11 cases of CDC (prevalence 1.9%) treated either by radical (9 cases) or partial nephrectomy (2 cases). During radical nephrectomy a loco-regional lymphadenectomy was always performed. In the 9 cases observed after 1997, a complete cytogenetic analysis of the neoplastic karyotype was carried out. RESULTS: At pathological examination the disease was found to be confined to the renal capsule (TNM 1997 stage 1) in only 3 patients; venous neoplastic trombosis and nodal metastasis were present in 3 and 6 cases respectively; 2 patients showed distant metastases (lung, bone). Two of the patients affected with stage 1 tumours are still alive with no evidence of the disease at 48 and 88 months after surgery, while the third died following the systemic progression of a concomitant bladder carcinoma. One patient with stage 4 tumour (no. 11) is alive, but the follow up time is still limited (2 months). All the other 7 patients are dead after a mean survival time of 16.3 months (range 0-45). As for cytogenetic analysis, 2 CDCs didn't grow in culture and in one case no karyotype alterations were reported. In the remaining 6 cases hypodiploid stemlines and a homogeneous chromosome alteration pattern were observed, with multiple numerical and structural aberrations (mean 11.1, range 7-15) and the continuous involvement of chromosomes 1 and X or Y, both as traslocation and deletion/monosomy. Additional abnormalities of chromosomes 22 and 13 were found to be common but less frequent. CONCLUSIONS: The clinical behaviour of the CDC is aggressive and its prognosis is surely poor; surgical treatment seems to be curative only for organ-confined cancer, accounting for the minority of cases. This neoplasm is cytogenetically characterized by hypodiploid stemlines with common involvement of chromosome 1 and the autosomes.

Dilated intercellular spaces as markers of reflux disease: histology, semiquantitative score and morphometry upon light microscopy.

BACKGROUND AND AIMS: A recent electron microscopy study suggested that dilated intercellular spaces (DIS) are specific for acid reflux-damaged esophageal epithelium. Electron microscopy is, however, expensive and difficult to apply to routine biopsies. The aims of this study are to establish a method for assessing DIS on light microscopy of esophageal biopsies and to estimate its association with current clinicopathological parameters of esophagitis. MATERIALS AND METHODS: 21 patients with reflux symptoms were investigated. Light microscopy biopsies were assessed for DIS size by a semiquantitative method and computer-assisted, static morphometry. A DIS score accounting for DIS size and distribution was assigned to each patient and its association with 30 clinicopathological variables investigated by univariate and multivariate logistic regression. RESULTS: Both the semiquantitative method and static morphometry identified 4 different classes of DIS size. The DIS score was significantly and independently associated with the esophageal symptoms score, the histological score of esophagitis and the relevant morphometry data. CONCLUSIONS: DIS may be efficiently assessed during light microscopy of routine esophageal biopsies. Since correlation with both the histology and the symptoms of esophagitis, the DIS score may be considered a novel parameter of esophagitis and is suggested for the routine evaluation of esophageal biopsies in patients with reflux disease.

p53 is dispensable for apoptosis but controls neurogenesis of mouse dentate gyrus cells following gamma-irradiation.

Mammalian cells respond to DNA insults by activating cell-cycle checkpoints. This may result in a temporary cell growth arrest which allows DNA repair before proliferation or induces apoptosis. p53 is one of the main contributors in regulating these activities. To get a better insight on the molecular mechanism underlying these activities we studied the role of p53 in apoptosis and neurogenesis of brain cells from adult p53(+/+) or p53(-/-) mice exposed to gamma-irradiation. Apoptosis and neurogenesis were assessed up to 14 days following the injury. Five-ten hours following gamma-irradiation, cells with TUNEL positive nuclei were identified within the subgranular zone of dentate gyrus (DG) of both p53(+/+) and p53(-/-) mice. At the same time-points, pyknotic and shrinking nuclei were visualized by Hoechst 33258 staining. Furthermore, gamma-irradiation increased the number of proliferating cell nuclear antigen (PCNA) positive cells with a peak at 5-10 h in both animal groups. PCNA immunoreactivity was detected in cells exhibiting condensed nuclei as visualized by Hoechst 33258 staining. Neurogenesis, assessed by mitotic marker p34(cdc2) immunoreactivity, showed a biphasic response to gamma-irradiation both in p53(+/+) and p53(-/-) mice which was characterized by an early inhibition and a delayed stimulation. In p53(-/-) mice, the time required by DG granule cells to recover from the lesion and to stimulate proliferation was significantly shortened in comparison with wild-type mice thus resulting in an accelerated neurogenesis. Our data indicate that following gamma-radiation p53 plays a role in regulating cell-cycle progression rate but it is dispensable for promoting apoptosis of DG granule cells.

Rectal localization of metastatic lobular breast cancer: report of a case.

The incidence of extrahepatic gastrointestinal metastases from breast cancer is reported in the literature only as necroscopy studies (6-18 percent); they usually originate from lobular or a mixed ductal-lobular subtype. Nonspecific presenting symptoms, death of the patients caused by other more frequent metastases, and variable radiographic features mimicking primary neoplasms cause a clinical underestimation of this pathology. We report here a case of rectal metastasis from a lobular carcinoma eight years after mastectomy.

High resolution allelotype of nonfunctional pancreatic endocrine tumors: identification of two molecular subgroups with clinical implications.

A high resolution allelotype for nonfunctional pancreatic endocrine tumors (NF-PETs) has been generated by microsatellite analysis of DNA from 16 frozen cases, each probed with 394 markers. Two subgroups of NF-PETs were found. Seven cases showed frequent, large allelic deletions [loss of heterozygosity (LOH)] with an average fractional allelic loss (FAL) of 0.55, whereas nine cases showed a small number of random losses with a FAL of 0.15. Designated high or low FAL, respectively, these genetic phenotypes showed correlation with the ploidy status: high-FAL tumors were aneuploid, low-FAL were diploid. Chromosomes 6q and 11q showed LOH in >60% of cases. About 50% of cases had losses on 11p, 20q, and 21. Selected LOH analysis on an additional 16 paraffin-embedded NF-PETs confirmed the high frequency of 6q and 11q LOH. The allelotype of NF-PET is markedly different from that of either ductal or acinar tumors of the pancreas as well as from that of functional-PETs. Moreover, whereas deletions involving chromosome 11 also are a feature of functional-PETs, the involvement of chromosome 6q is characteristic of NF-PETs. Survival analysis showed that none of the single chromosomal alterations was associated with outcome, whereas ploidy status is an independent factor adding prognostic information to that furnished by the proliferative index measured by Ki-67 immunohistochemistry.

Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-alpha.

BACKGROUND: Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS: We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS: Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

The spleen in the Wiskott-Aldrich syndrome: histopathologic abnormalities of the white pulp correlate with the clinical phenotype of the disease.

The Wiskott-Aldrich syndrome (WAS) is a X-linked hematologic disorder characterized by thrombocytopenia, eczema, and immunodeficiency of variable severity. Reported here are the results of a morphologic, morphometric, and immunophenotypic analysis of splenic lymphoid tissue in 12 WAS patients with documented molecular defect and with different disease severity. Spleens from 29 age-matched patients with different diseases were used as controls. Paraffin-embedded tissue (from all cases) and fresh-frozen samples (from 5 WAS patients and 4 control subjects) were used to study the different white pulp compartments by classic morphologic, immunophenotyping, and image analysis techniques. Data were statistically analyzed by both parametric and nonparametric tests. Spleens from WAS patients showed a significant depletion of the total white pulp (p = 0.0008), T cell (p < 0.05), and B cell (p = 0.0002) areas and marginal zone (MZ) thickness (p < 0.0001). Among WAS patients, a negative correlation was found between the score of severity of the disease and all variables considered (Spearman's rank correlation coefficient, r = -0.79, r = -0.73, r = -0.68, and r = -0.56, respectively). In conclusion, this study shows that in WAS a general depletion of the splenic white pulp occurs, supporting the evidence that WAS is characterized by a combined immune defect. The significant reduction of the MZ may explain the inability of WAS patients to mount a response to T-independent antigens.

Multitechnical pathological diagnosis in chromophobe renal cell carcinoma.

Two new cases of chromophobe renal cell carcinoma were diagnosed on the basis of their morphology and their karyotype complemented by flow cytometry. In one of these cases, however, all these investigations were not sufficient and additional histochemistry investigation had to be used to completely rule out other renal tumors such as oncocytoma, the prognosis of which is totally different.

[Flow cytometry sutdy of DNA and cell kinetics in the adenoma- carcinoma sequence in the large intestine]. / Studio citofluorimetrico del DNA e della cinetica cellulare nella sequenza adenoma-carcinoma del grosso intestino.

UNLABELLED: The quantitation of DNA and growth fraction in the different step from dysplastic to neoplastic process in large bowel is the aim of this study. 70 colonic polyps were studied. The fresh specimens were processed and DNA analysis was carried out using a Partec CA II flow cytometer and the growth fraction was tested with KI-67 monoclonal antibody. The percentage of S-phase cells has been calculated with the Multicycle program. Our results demonstrated that 7 adenomas were tubulo-villous with mild dysplasia, 39 with mild-moderate dysplasia, 1 with severe dysplasia, 5 were polypoid carcinomas, 2 juvenile polyps, 1 polypoid leiomyoma, 1 inflammatory fibroid polyps. DNA analysis showed a diploid DNA content in non adenomatous polyps, in all adenomas with mild dysplasia, in 37 with mild-moderate dysplasia, in 8 cases with moderate-severe dysplasia and 1 cancer. Aneuploidy was discovered in 2 cases with mild-moderate dysplasia, in 6 cases with moderate-severe dysplasia, in the case of severe dysplasia and in 4 cases of carcinomas. Best indexes of linear correlation (Pearson's r) has been found between S-phase and DNA index (r = .75) and between S-phase and KI-67 (r = .82). IN CONCLUSION: 1) No relationship was found between DNA content and age, sex, size and location of polyps. 2) Aneuploidy is strictly related to moderate-severe grade of dysplasia therefore it is an important element in the development of adenomacarcinoma sequence. 3) DNA-index, S-phase and KI-67 are strictly related.

HLA class I antigen and transporter associated with antigen processing (TAP1 and TAP2) down-regulation in high-grade primary breast carcinoma lesions.

Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for expression of HLA antigens and components of the antigen-processing machinery by immunohistochemical staining. The expression of transporter associated with antigen processing (TAP) 1, TAP2, and HLA class I antigens in breast carcinoma lesions was significantly associated with tumor grading. Like normal mammary tissue, the 16 low-grade (G1) breast carcinoma lesions showed strong staining for TAP1, TAP2, and HLA class I antigens. In contrast, only 12 (32%) of 37 high-grade (G2 and G3) breast carcinoma lesions displayed the normal staining pattern. In 14 (38%) of 37 high-grade lesions, HLA class I antigen down-regulation was observed without loss of low molecular mass polypeptide and/or TAP staining. Congruent down-regulation of HLA class I antigen and TAP1 or TAP2 was found in 8 (22%) of 37 high-grade lesions. Complete loss of HLA class I antigens, TAP1, and TAP2 was observed in 3 (8%) of 37 high-grade lesions. No lesion was negative for TAP1 and/or TAP2 staining while positive for HLA class I antigen staining. These data demonstrate an association of HLA class I antigen and TAP down-regulation with tumor progression in breast carcinoma. This association suggests that loss of HLA and/or TAP may represent an escape from the host's immune pressure or may reflect the accumulation of abnormalities associated with neoplastic progression. This accumulation of defects in antigen processing and presentation may in turn be responsible for reduced recognition of malignant cells by putative clinically relevant tumor-specific T cells.