Histological insights into the pathogenesis of post-Roux-en-Y hyperinsulinaemic hypoglycaemia.

BACKGROUND: ß-cell hyperplasia has been implicated in the aetiology of post Roux-en-Y gastric bypass hyperinsulinaemic hypoglycaemia, but the pathogenesis of this condition is still unclear. CASE REPORT: We report a case of a 52-year-old man with post-Roux-en-Y gastric bypass hyperinsulinaemic hypoglycaemia who underwent distal pancreatectomy to alleviate his symptoms. Pancreatic histopathology showed chronic pancreatitis with a corresponding loss of exocrine tissue and islet retention. Amyloid deposition was found in pancreatic islets. These features are more typically associated with Type 2 diabetes. DISCUSSION: This case highlights the potential multifactorial pathogenesis of symptomatic hypoglycaemia after Roux-en-Y gastric bypass.

TTF1 expression in non-small cell lung carcinoma: association with TTF1 gene amplification and improved survival.

Acquired chromosomal aberrations play an important role in tumour development and progression. Such genetic alterations occur in a significant proportion of non-small cell lung carcinomas (NSCLCs) and include amplification of 14q13.3, which contains the TTF1 gene. We asked whether TTF1 amplification is associated with increased TTF1 protein expression in NSCLCs, and whether TTF1 is associated with clinicopathological features, including patient survival. We used a FISH assay and quantitative immunohistochemical staining to interrogate a population-based cohort of 538 NSCLCs from Swiss patients for TTF1 amplification and protein expression. We found TTF1 amplification in approximately 13% of adenocarcinomas (ACs) and in approximately 9% of squamous cell carcinomas (SCCs) and TTF1 amplification was associated with increased TTF1 protein expression. High-level TTF1 expression was significantly associated with smaller tumour size, female gender and longer overall survival only among ACs (median survival 82 versus 28 months; p = 0.002). On multivariate analysis, high TTF1 expression was an independent predictor of favourable prognosis in patients with AC [hazard ratio, 0.56 (95% CI 0.38-0.83); p = 0.008]. We conclude that TTF1 amplification is a mechanism of high-level TTF1 expression in a subset of NSCLCs. When expressed at high levels, this routinely used diagnostic marker is also an independent biomarker of favourable prognosis in AC.

Transcriptome analysis of endocrine tumors: clinical perspectives.

There is considerable interest in the application of DNA microarrays to the pathologic evaluation of endocrine neoplasms. Improvements in tumor classification and prognostication, prediction of response to therapy, and comprehensive assessment of tumoral hormone production represent the major anticipated benefits. Here, some of the microarray studies that support the clinical use of transcriptome profiling for endocrine tumors are reviewed. In addition, some of the barriers to clinical implementation are discussed.

Genomic amplification of MET with boundaries within fragile site FRA7G and upregulation of MET pathways in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) is characterized by a poor prognosis making the identification of clinically targetable proteins essential for improving patient outcome. We report the involvement of multiple alterations of the MET pathway in EA development and progression. Microarray analysis of Barrett's metaplasia, dysplasia, and EA revealed overexpression of the MET oncogene in EAs but only those with MET gene amplification. STS-amplification mapping revealed that the boundary of the MET amplicon in these EAs is defined by fragile site FRA7G. We also identified an amplicon at 11p13 that resulted in amplification and overexpression of CD44, a gene involved in MET autophosphorylation upon HGF stimulation. Tissue microarrays with phospho-MET-specific antibodies demonstrated a uniformly high abundance of MET activation in primary EA and cells metastatic to lymph nodes but to a lesser extent in a subset of metaplastic and dysplastic Barrett's samples. Increased expression of multiple genes in the MET pathway associated with invasive growth, for example, many MMPs and osteopontin, also was found in EAs. Treatment of EA-derived cell lines with geldanamycin, an inhibitor for tyrosine kinases including MET receptor kinase, reduced cell migration and induced EA cell apoptosis. The data indicate that upregulation of the MET pathway may contribute to the poor outcome of EA patients and that therapeutic agents targeting this pathway may help improve patient survival.

Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.

We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.

Management of patients with adrenal cancer: recommendations of an international consensus conference.

Adrenocortical carcinomas are rare, highly malignant tumors that account for only 0.2% of deaths due to cancer. Given the limited number of patients seen in most medical centers with this diagnosis, series usually reported are small and clinical trials not randomized or blinded. In an attempt to answer important questions concerning the management of patients with adrenal cancer, a consensus conference was organized and held at the University of Michigan in Ann Arbor, MI, 11-13 September 2003, with the participation of an international group of physicians who had reported on the largest series of patients with this disease and who had recognized basic and clinical research expertise in adrenal cortical cancer. Totally 43 questions were addressed by the presenters and recommendations discussed in plenary and breakout sessions. Evidence for the recommendations of this conference was at the 2-4+ level and based on available literature and participants' experience. In addition to setting up guidelines in specific areas of the diagnosis and treatment of adrenal cancer, the conference recommended and initiated the planning of an international prospective trial for treatment of patients with adrenal cancer in stages III and IV. In terms of new therapies, first trials of dendritic cell therapy in human subjects with adrenal cancer have been started, but it is too early to comment on efficacy. Different strategies of immunotherapy, including DNA vaccination are currently being tried in animal models. There are no clinical gene therapy trials for human adrenal cortical cancer. The adrenals are a preferred target for adenovirus and the results of gene therapy in preclinical studies are promising. In addition, there is evidence that histone deacetylase inhibitors can further enhance the rate of adenoviral infectivity in human adrenal cancer cells. Testing of retroviral vectors, non-viral vectors, small interfering RNA technology, and combined approaches could be performed in various laboratories. Anti-angiogenic substances have only been applied in preclinical studies. The use of these and other agents in the treatment of adrenal cancer should be hypothesis-driven and based on a thorough analysis of tumor biology.

Inflammatory cytokine regulation of TRAIL-mediated apoptosis in thyroid epithelial cells.

Death receptor-mediated apoptosis has been implicated in target organ destruction in chronic autoimmune thyroiditis. Depending on the circumstances, inflammatory cytokines such as IL-1, TNF and IFNgamma have been shown to contribute to either the induction, progression or inhibition of this disease. Here we demonstrate that the death ligand TRAIL can induce apoptosis in primary, normal, thyroid epithelial cells under physiologically relevant conditions, specifically, treatment with the combination of inflammatory cytokines IL-1beta and TNFalpha. In contrast, IFNgamma is capable of blocking TRAIL-induced apoptosis in these cells. This regulation of TRAIL-mediated apoptosis by inflammatory cytokines appears to be due to alterations of cell surface expression of TRAIL receptor DR5 and not DR4. We also show the in vivo presence of TRAIL and TRAIL receptors DR5 and DcR1 in both normal and inflamed thyroids. Our data suggests TRAIL-mediated apoptosis may contribute to target organ destruction in chronic autoimmune thyroiditis.

Proteomics-based identification of RS/DJ-1 as a novel circulating tumor antigen in breast cancer.

PURPOSE: We used a proteomics-based approach to identify tumor proteins that elicit a humoral response in breast carcinoma and that may occur as circulating antigens. EXPERIMENTAL DESIGN: The breast cell line SUM-44 was used as a source of tumor cell proteins for two-dimensional PAGE (2-D PAGE) and for Western blot analysis in which individual sera were analyzed for primary antibodies. RESULTS: Sera from 30 newly diagnosed patients with breast cancer were screened for IgG antibodies to tumor cell proteins. Sera from 116 patients with other cancers and from 25 healthy subjects served as controls. Restricted reactivity against a set of three proteins, identified by mass spectrometry as isoforms of a novel oncogenic protein that regulates RNA-protein interaction (designated RS/DJ-1), was observed in four patients with breast cancer, but not in healthy subjects. The identity was further confirmed by Western blotting with specific antibodies. RS/DJ-1 was found to be secreted in the breast cell line SUM-44, which led us to determine whether RS/DJ-1 was found in circulation in breast cancer. Interestingly, unlike in controls, RS/DJ-1 was readily detectable in sera from 37% of newly diagnosed patients with breast cancer. CONCLUSION: The presence of autoantibodies and/or circulating RS/DJ-1 protein in sera from patients with breast cancer may have clinical utility.

1Alpha,25-dihydroxyvitamin D3 up-regulates Bcl-2 expression and protects normal human thyrocytes from programmed cell death.

Apoptosis is thought to play an important role in the pathogenesis of autoimmune thyroid disease. 1alpha,25-dihydroxyvitamin D3 (VD3) has been shown to suppress several autoimmune diseases. However, the mechanism by which VD3 has these effects is not known. We evaluated the alterations in apoptosis, induced by VD3. Thyrocytes were treated with VD3, and the expression of the Bcl-2 family molecules was studied at both the messenger RNA and protein levels. It was found that VD3 significantly induced the expression of Bcl-2 messenger RNA and protein in thyrocytes but had no effect on the expression of Bcl-xl and Bax. The increase in Bcl-2 expression, mediated by VD3, correlated with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation. VD3-induced increases in the expression of Bcl-2 could be mimicked by VD3 analogs with high nuclear receptor affinity, but not by analogs only with nongenomic actions. These data indicate a role for Bcl-2 in the regulation of apoptosis in thyrocytes and raise the possibility that VD3 or its agonists may have therapeutic benefit in thyroid disorders.

Fas (CD95) expression is up-regulated on papillary thyroid carcinoma.

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma and cycloheximide. Whereas both normal and cancerous thyrocytes were induced to die after this treatment, the cancerous thyrocytes were more sensitive to anti-Fas antibody. This work demonstrates that the Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.

Overexpression of CXC chemokines by an adrenocortical carcinoma: a novel clinical syndrome.

A patient with adrenocortical carcinoma presented with fever, leukocytosis, and increased acute phase reactants. The tumor was infiltrated with neutrophils. Immunohistochemical staining of the tumor showed positive signal for epithelial neutrophil-activating protein-78, an angiogenic and chemotactic CXC chemokine. Conditioned medium from tumor-derived cells (RL-251) showed high concentration of IL-8, epithelial neutrophil-activating protein-78, Gro alpha, and Gro gamma, angiogenic CXC chemokines with a potential role in tumorigenesis. An adrenal cancer/severe combined immunodeficiency mouse chimera was developed. Mice grew tumors rapidly, and circulating levels of IL-8 and epithelial neutrophil-activating protein-78 were detected. In contrast, animals transplanted with NCI-H295 cells, a nonchemokine-secreting cell line, grew tumors more slowly and did not have detectable chemokine levels. Similar to the patient, mice with RL-251 tumors developed marked leukocytosis and neutrophilia, and their tumors were infiltrated with neutrophils. Mice were passively immunized with epithelial neutrophil-activating protein-78 antisera. A marked decrease in tumor growth was observed. Potential for chemokine production by other adrenocortical tumors was investigated by RT-PCR in archival material. Six of seven adrenal carcinomas and one of three adenomas had cDNA for IL-8; six of seven carcinomas and the three adenomas had cDNA for epithelial neutrophil-activating protein-78. We concluded that the clinical presentation of this case resulted from increased tumor production of chemotactic chemokines. Through their angiogenic and chemotactic properties these chemokines may play an important role in adrenal tumorigenesis.

Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells.

The HyR gene, conferring resistance to hygromycin B (Hy), has been modified for optimal expression in mammalian cells. Modifications to the HyR gene and its expression cassette include: (1) removal of all upstream start codons, (2) conversion of the region around the start codon to the consensus sequence associated with efficient translation initiation, and (3) removal of downstream splice donor and acceptor sequences. The resulting HyR gene is an efficient dominant selectable marker that is useful for studies requiring resistance from a low-copy-number gene driven by a promoter of moderate strength. The HyR gene was also tested for its compatibility with BPV vectors. Mouse C127 cells harboring pHyR-BPV plasmids exhibited properties of BPV-transformed cells and were resistant to toxic levels of Hy. The vectors were stable as episomes and present in high copy. The HyR gene thus joins the NmR (neo) gene as the only dominant selectable markers that are known to be compatible with BPV replication.

Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system.

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.

Comparison of genetic alterations in colonic adenoma and ulcerative colitis-associated dysplasia and carcinoma.

Carcinoma is an important complication of ulcerative colitis (UC) and develops from dysplastic precursor lesions. Genetic changes involved in the malignant transformation have not been fully characterized. We studied 19 cases of UC with high-grade dysplasia (HGD) and eight samples of associated carcinoma (CA). Microdissection of normal epithelium, epithelium at the site of chronic inflammation, HGD, and CA was performed. Polymerase chain reaction (PCR) amplification for loss of heterozygosity (LOH) of the following polymorphic microsatellites of putative tumor suppressor gene loci was done: APC (5q), DCC (18q), p16 (9p), p53 (17p), and 8p12. To compare genetic alterations, 22 typical adenomas of the colon were studied with the markers for APC and pl6 gene loci. The results indicated that LOH of p16 and p53 were present in nondysplastic epithelium, HGD, and CA. However, the LOH in nondysplastic epithelium was detected in some associated HGD, but not all. Whereas LOH of p16 was present in 7 of 14 cases of HGD (50%), it was noted in only 1 of 22 adenomas (5.0%). LOH in the APC and DCC gene loci in UC was noted in HGD with associated CA, but LOH of APC was not present either in cases of nondysplastic epithelium or in HGD alone. Conversely, LOH in APC was present in 4 of 19 colonic adenomas. We conclude that LOH of p53 and p16 in nondysplastic epithelium may be associated with chronic reparative processes. These changes may lead to susceptibility to further genetic damage involving the APC and DCC gene loci in the development of dysplasia and progression of CA in UC. The low frequency of LOH in the p16 gene (9p) in adenomas compared with dysplasia in UC combined with infrequent LOH in APC gene loci in cases of pure dysplasia in UC may support this combination of markers as a clinical test for the differentiation of polypoid dysplasia from adenomas in UC.

Comparison of intraoperative cytology with frozen sections in the diagnosis of thyroid lesions.

We retrospectively studied the usefulness of intraoperative cytology (IOC) and frozen section (FS) in the rapid diagnosis of 68 thyroid lesions. In 14 cases of papillary thyroid carcinoma, IOC correctly diagnosed 13 cases, while FS correctly diagnosed 11 cases. There was no significant difference in sensitivities, and both methods had similar specificities. In 21 cases of colloid nodule, IOC was slightly more sensitive than FS; IOC correctly diagnosed 16 cases, while FS correctly diagnosed 15 cases. However, the specificity of IOC was only 71%, but was 98% for FS. Of 17 follicular adenomas, FS diagnosed 16 as follicular neoplasms and misdiagnosed only 1 as a colloid nodule. By contrast, IOC misdiagnosed 9 follicular adenomas as colloid nodules, most of which were macrofollicular variants with abundant colloid. Of 11 follicular carcinomas, FS diagnosed all as follicular neoplasms, while IOC misdiagnosed 3 as colloid nodules. While IOC is not as accurate as FS in the diagnosis of colloid nodules and follicular neoplasms, it is highly sensitive and specific in the diagnoses of papillary carcinoma and performance of the technique is rapid and easy. In an intraoperative setting, IOC is a useful adjunct to FS in screening thyroid nodules for the presence of papillary carcinoma.

Serum thyroglobulin levels after thyroxine withdrawal in patients with low risk papillary thyroid carcinoma.

We hypothesized that elevated levels of serum thyroglobulin (Tg) are frequently found as the only index of residual neoplasm in patients with low-risk papillary thyroid carcinoma. The records of patients operated on for papillary thyroid carcinoma over a 2-year period were reviewed, and the patients were allocated to risk groups by a validated staging method that does not include Tg levels. Of the 35 patients who manifested a low-risk carcinoma, 9 (26%) exhibited elevated Tg concentrations (11-53 ng/mL) during thyroxine withdrawal after therapies, while clinical, scintigraphic, and radiographic studies at least 1 year later showed no evidence of tumor. Prior scintigraphic imaging of therapeutic doses of 131I in 8 of 9 patients demonstrated no distant metastases, further confirming the low-risk status of this group. The staging method predicts that only 0.9% of patients with low-risk papillary carcinoma will have a cause specific death in 20 years. Elevated Tg concentrations have not been shown to forecast independently the survival of patients with low-risk papillary carcinoma. Thus, although frequently encountered, elevated Tg concentrations are unlikely to predict shortened survival in patients with papillary carcinoma for whom low risk has been determined from other data.

Treatment of micronodular lung metastases of papillary thyroid cancer: are the tumors too small for effective irradiation from radioiodine?

Our purpose was to determine if micronodular lung metastases from papillary thyroid cancer had diameters that were less than 1 mm and therefore of a size for which irradiation by radioiodine (131I) is inefficient. In five patients, lung metastases seen on computed tomography (CT) were enumerated and sized in the entire right lung and right upper lung giving volumes of measurable, ie, more than 1 mm diameter, tumors. Concentrations of diagnostic 131I were quantified scintigraphically in the same regions. Fractions of administered 131I per milliliter of tumor and the absorbed radiation from the subsequent treatments were calculated to see if the 131I levels in lungs were greater than expected for the visible tumor volumes. Two other patients manifesting similar findings had lung biopsies that were reviewed for size of metastases. The calculated fractions of administered activity per milliliter of tumor and the absorbed radiations from the treatments with 131I were exceptionally high. Biopsies revealed numerous tumors below the resolution of CT. We conclude that the fractions of administered activity and absorbed radiations of 131I in tumors were high because the measured tumor volumes underestimated the total tumor volumes. Many lung metastases were less than 1 mm in diameter.

Detection and speciation of mycobacteria in formalin-fixed, paraffin-embedded tissue sections.

The ability to detect mycobacterial DNA by polymerase chain reaction (PCR)-based methodology in formalin-fixed, paraffin-embedded tissue sections is useful in several clinical scenarios. The major use of this type of assay is in those instances in which infectious disease is not clinically suspected and microbiological cultures are not performed. In these cases, the only tissue available for examination is that present in routinely prepared paraffin blocks after histologic examination. The presence of necrotizing granulomatous inflammation should result in special stains for acid-fast organisms. However, in many such cases the special stains are unsatisfactory, because the number of organisms present is very low (as in Mycobacterium tuberculosis). Thus, the tedious examination of multiple serial sections is often necessary to identify the pathogenic organism, and often no organism is found. Therefore, more sensitive detection methods are needed. PCR-based detection of mycobacterial DNA is more sensitive and can be used either to verify the presence of organisms seen on special stains or to identify an occult organism. By combining the PCR assays with restriction analyses of the products, it is often possible to speciate the pathogenic organism.

Granular-cell tumor of trachea masquerading as Hurthle-cell neoplasm on fine-needle aspirate: a case report.

We report on a case of extraluminal tracheal granular-cell tumor which was interpreted as a Hurthle-cell neoplasm of the thyroid on fine-needle aspirate. Review of the literature reveals only one other such case. The patient was a 35-yr-old female who presented with an enlarged thyroid. Aspiration cytology revealed a syncytium of cells with abundant granular cytoplasm interpreted as a thyroid follicular neoplasm with Hurthle-cell change. However, histology of the resection specimen with immunohistochemistry confirmed it as a granular-cell tumor. The cytologic differential diagnosis of neoplasms with oncocytoid cytoplasm in and around the thyroid should include granular-cell tumor of the trachea.