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Gastric fundic gland polyps (FGPs) are considered hamartomas, and various gastrointestinal hamartomas are associated with tuberous sclerosis complex (TSC). The aim of this study was to investigate a possible link between TSC proteins (hamartin and tuberin) and sporadic FGPs. We examined 33 sporadic FGPs and 26 biopsies of normal fundic mucosa by immunohistochemistry. Nuclear immunoreactivity for tuberin was dramatically reduced or lost in most sporadic FGPs, and tuberin unexpectedly accumulated in the cytoplasm in oxyntic glands. About 18% (6/33) of FGPs were immunopositive in an average of 1.7% of oxyntic cell nuclei, compared with 77% (20/26) of controls in an average of 24.4% of oxyntic cell nuclei (P

The IR microspectra for individual normal and cancerous cells are reported at a spatial resolution that permits a distinction between the nuclear and cytoplasmic regions. The observed spectra reveal large differences in the spectral contributions of RNA, DNA, and phospholipids: metabolically inactive cells show spectral patterns of proteins only, whereas actively dividing cells also show spectral signatures of nucleic acids and phospholipids. These spectral variations are independent of the state of health of a cell.

Tuberous sclerosis complex (TSC) is a genetically heterogeneous disease caused by mutations of TSC1 or TSC2 genes. It involves multiple organ systems resulting in mild to lethal hamartoma formation due to gene mutation in the germ line and loss of heterozygosity (LOH) in somatic cells. Hamartin ( TSC1) and tuberin ( TSC2) are expressed broadly. However, little is known about tissue susceptibility to hamartomas when equal or similar amounts of TSC gene expression are present. In this study, we present a 19-week gestational age fetus with pathological features of TSC, which was confirmed by finding LOH of TSC2 in a cardiac rhabdomyoma. Developmental expression of hamartin and tuberin in the TSC fetus, an age-matched non-TSC fetus, and a 26-week gestational age non-TSC fetus were analyzed by immunohistochemistry. We found that in addition to the differential expression of the TSC genes in some normal tissues compared with that in the TSC-affected fetus, the cellular localization and distribution of hamartin and tuberin were dramatically different in different tissues. In general, hamartin and tuberin are mainly expressed in epithelial cells, myocytes, and neural tissues. By comparing the incidence of the hamartomas in early childhood and gene expression in tissues, it appears that tissues with co-expression of hamartin and tuberin are prone to a higher incidence of hamartomas than those expressing only one protein, or two proteins but in different patterns of cellular localization.

BACKGROUND The diagnosis of melanoma can be difficult because of shared cytomorphology with other malignant neoplasms. The most commonly used melanocytic markers, anti-S-100 protein and HMB-45 antigen, have limited specificity and sensitivity, respectively. Microphthalmia transcription factor (Mitf) is a nuclear transcription factor critical for the development and survival of melanocytes and has been shown as a sensitive and specific marker for melanoma in histologic specimens. METHODS To evaluate the efficacy of Mitf as a marker for melanoma in cytologic preparations, 81 cell blocks from 44 patients with melanoma and 37 patients with nonmelanoma malignancies (29 patients with carcinoma, 4 patients with mesotheliomas, 2 patients with lymphoma, and 2 patients with islet cell tumors) were stained with monoclonal antibodies against Mitf (clone D5), S-100 protein, and HMB-45 antigen. The staining was evaluated blindly by three independent observers. The presence of nuclear staining for Mitf and cytoplasmic staining for S-100 protein or HMB-45 antigen in > 10% of tumor cells was considered positive staining for each antigen. RESULTS Forty-four melanomas (100%), including all 3 spindle-cell melanomas, were positive for Mitf. All nonmelanoma neoplasms were negative with only one exception: One mammary carcinoma showed rare (< 10%), weak nuclear staining with Mitf. The sensitivity and specificity of Mitf as a marker for melanoma were both 100%, whereas the sensitivity of HMB-45 antigen was 90.4%, and the specificity of S-100 protein was 70.3%. CONCLUSIONS Mitf is a sensitive and specific marker for malignant melanoma, including the spindle-cell variant, in cytologic specimens and may be superior to the current standard melanocytic markers, S-100 protein and HMB-45 antigen. Cancer (Cancer Cytopathol) 2001;93:337–343. © 2001 American Cancer Society.

Hypothesis Increased cell motility is a hallmark of cancer cells. Proteins involved in cell motility may be used as molecular markers to characterize the malignant potential of tumors. Methods Molecular biology and immunohistochemistry techniques were used to investigate the expression of a selected panel of motility-related proteins (Rho A, Rac 2, Cdc42, PI(3)K, 2E4, and Arp2) in normal, premalignant, and squamous cell cancer cell lines of human head and neck origin. To assess the clinical potential of these proteins as molecular markers for cancer, immunohistochemistry was performed on paraffin-fixed head and neck cancer specimens (n = 15). Results All six motility-associated proteins were overexpressed in the premalignant and squamous cell cancer cell lines relative to normal keratinocytes. Immunohistochemistry with Rho A and Rac 2 showed increased staining in areas of cancer but not in normal tissue. Conclusion Proteins involved in cell motility can be used as markers for head and neck squamous cell carcinoma. The head and neck cell lines used in this study may be used as a model to further investigate cell motility. Molecular markers of motility could have a significant impact on the diagnosis and staging of cancers originating from differentiated non-motile cells.

Small, extraportal, hepatic parenchymal cells, positive for biliary-type cytokeratins, may represent hepatic stem cells, canals of Hering (CoH), and/or ductal plate remnants. We evaluated these cells 3 dimensionally in normal human liver and massive necrosis. Tissues from normal human livers and from 1 liver with acetaminophen-induced massive necrosis were serially sectioned, immunostained for cytokeratin 19 (CK19), and sequentially photographed. Images were examined to determine 3-dimensional relationships among CK19-positive cells. Immunostains for other hepatocyte and progenitor cell markers were examined. In normal livers, intraparenchymal CK19-positive cells lined up as linear arrays in sequential levels. One hundred of 106 (94.3%) defined, complete arrays within levels examined, most having 1 terminus at a bile duct, the other in the lobule, beyond the limiting plate. In massive necrosis, there were 767 individual CK19-positive cells or clusters around a single portal tract, 747 (97.4%) of which were spatially related forming arborizing networks connected to the interlobular bile duct by single tributaries. C-kit was positive in normal CoH. CK19 co-expressed with HepPar1, c-kit, and alpha-fetoprotein (AFP) in parenchymal cells in massive necrosis. Small, extraportal, biliary-type parenchymal cells represent cross-sections of the CoH that radiate from the portal tract, usually extending past the limiting plate into the proximate third of the hepatic lobule. The 3-dimensional structure of ductular reactions in massive necrosis suggests that these reactions are proliferations of the cells lining the CoH. Therefore, the CoH consist of, or harbor, facultative hepatic stem cells in humans.

This contribution is aimed at introducing a new spectroscopic endeavor, infrared spec-troscopy of entire cells and tissues, and demonstrating that this field has enormous potential as a diagnostic tool in medicine and cell biology.

Allele and genotype frequencies were determined for the HLA-DQA1 and Amplitype® Polymarker loci (low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group-specific component (Gc)) in Hasidic and non-Hasidic Ashkenazi New York City Jewish subpopulations. For all loci tested, except HBGG, the 2 subpopulations meet the assumption of Hardy-Weinberg equilibrium. Comparison of various allele and genotype frequencies for the Hasidic and the non-Hasidic groups showed no significant differences. Comparison of the various allele frequencies in the two subpopulations to another Caucasian group revealed significant differences at the HLA-DQA1 and D7S8 loci in the Hasidic group. These frequency data can be used for comparison to other populations and for frequency estimates in DNA profiling.

Background Young breast cancer (BC) patients less than 45 years old are at higher risk of dying from the disease when compared to their older counterparts. However, specific risk factors leading to this poorer outcome have not been identified. Methods One candidate is iron deficiency, as this is common in young women and a clinical feature of young age. In the present study, we used immuno-competent and immuno-deficient mouse xenograft models as well as hemoglobin as a marker of iron status in young BC patients to demonstrate whether host iron deficiency plays a pro-metastatic role. Results We showed that mice fed an iron-deficient diet had significantly higher tumor volumes and lung metastasis compared to those fed normal iron diets. Iron deficiency mainly altered Notch but not TGF-β and Wnt signaling in the primary tumor, leading to the activation of epithelial mesenchymal transition (EMT). This was revealed by increased expression of Snai1 and decreased expression of E-cadherin. Importantly, correcting iron deficiency by iron therapy reduced primary tumor volume, lung metastasis, and reversed EMT markers in mice. Furthermore, we found that mild iron deficiency was significantly associated with lymph node invasion in young BC patients (p Conclusions Together, our finding indicates that host iron deficiency could be a contributor of poor prognosis in young BC patients.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

8545 Purpose: This clinical trial evaluates the safety and adjuvant activity of imiquimod, a toll-like receptor (TLR)-7 agonist, when given with a NY-ESO-1 protein vaccine. Imiquimod, by locally activating and recruiting dendritic cells (DCs) into the skin, is expected to stimulate antigen uptake by DCs, induce maturation and migration to draining lymph nodes, and to induce antigen-specific T and B cell immunity. Methods: Pilot study; 9 patients with resected stage 2B-3C malignant melanoma. Four 21 day cycles consisted of topical imiquimod cream (250 mg) on days 1–5 and id. injected NY-ESO-1 protein (100 mcg) into the site on day 3. Blood was drawn at several time points for immune monitoring; skin punch biopsies were obtained from control, imiquimod and vaccination sites 48 hours after the last vaccination. Results: The regimen was tolerated well, all patients completed four vaccinations. AEs were mild and transient and included injection site reactions (8/9 patients), fatigue (4/9 patients) and fever (2/9 patients). Significant levels of antigen-specific CD4+ or CD8+ T cell responses were not detected in ex-vivo ELISPOT assays. However, intracellular cytokine staining assays after in vitro pre-stimulation indicated that 6 of 8 subjects developed NY-ESO-1 CD4+ T cell responses. Humoral immunity was manifest by the induction of anti-NY-ESO-1 antibodies in 7/9 patients post-vaccination. Histochemistry of skin sections showed significant dermal mononuclear cell infiltrates in Imiquimod treated skin, whereas none were seen in untreated skin (p No significant financial relationships to disclose.

DNA was isolated from casework urine samples previously submitted for toxicological analysis. The quality and quantity of DNA isolated was determined by spectrofluorometry and agarose yield gel electrophoresis. Hae III restricted samples were then resolved by analytical agarose gel electrophoresis, transferred to a membrane by Southern blotting and hybridized with a chemiluminescently-labelled (D2S44) probe. The DNA fragment banding patterns were indistinguishable from the DNA banding patterns of blood specimens collected from the same donor. Only 5 of 20 samples yielded banding patterns and the banding intensity relative to background was low. Genomic DNA was also obtained from casework samples by Chelex extraction, amplified by polymerase chain reaction (PCR) and then genotyped for human leucocyte antigen (HLA) DQα. Of 20 specimens, 13 (65%) were typed correctly producing identical results for urine and blood specimens obtained from the same donor. Aging studies of casework samples and normal samples (from a non-drug using population) were also conducted with PCR-HLA DQα analysis. Results of these studies indicate that amplification by PCR was more likely to produce positive results. Based on these findings, we conclude that PCR-initiated analysis is more suitable than RFLP analysis for individualization of urine samples.

The diagnosis ofprostate cancer is based on the visible microscopic evaluation of both cytological and architectural featuresof the prostate tissue sections. In order to determine whether infrared (IR) spectral "mapping" can be used to objectivelydistinguish between normal and neoplastic prostate tissue, a comparison between "visual, point-by-point" and "automated,point-by-point" infrared measurements was perfonned. Automated, point-by-point analysis was performed without any prior diagnostic information. Visual, point-by-point measurements were based on histopathology, histochemistry and immunohistochemical analysis of the tissue samples. The spectra obtained from these measurements were compared to the spectra obtained from automated point-by-point analysis. Our results indicate that the spectra obtained from histopathologically directed measurements compares well with those of automated mapping methods. Therefore. we believethat current mapping methodology can be directly correlated with pathological diagnoses.Keywords: Infrared Microspectroscopy, Infrared Mapping, Histopathology. Prostate, Adenocarcinoma

Cutaneous patch and plaque lesions, and erythroderma may suggest mycosis fungoides both clinically and histopathologically in HIV+ patients. However, in some cases, this diagnosis is questionable. Five such cases are presented. When we compared these cases with cases of mycosis fungoides unassociated with HIV infection, we found less concordance among dermatopathologists in making a histopathological diagnosis, a greater proportion of CD8 than CD4 T cells in the cutaneous infiltrates, and no instances of demonstrated clonality of the cutaneous T-cell infiltrates in the HIV+ group. We conclude that CD8 T-cell predominant dermatoses may simulate mycosis fungoides in HIV+ patients.

In vivo animal models of primary brain tumors are necessary to advance knowledge related to the complex interactions between glioma cells and the adjacent brain. A cardinal feature of glioma growth, and a major reason why neurosurgical and adjunctive therapies ultimately fail in most patients is their invasive properties. We have adapted a previously described animal model developed by one of us to give better histological detail while preserving the identification of single infiltrating glioma cells. GL261 glioma cells were first transfected with the plasmid encoding green fluorescent protein (GFP) and then implanted into the brains of syngeneic C57BL/6 mice. Identification of GFP-positive tumor cells in paraffin sections of the brains of tumor-bearing animals utilized an antibody for conventional immunoperoxidase immunohistochemistry. This method is a more powerful technique compared with the prior use of frozen sections and fluorescence microscopy to identify GFP-tagged tumor cells. We find that this new method provides improved morphology and proves to be a sensitive and reliable system for detection of invading glioma cells. Using this methodology with other advanced technologies (eg, laser capture microdissection) holds out the promise of helping to elucidate the molecular mechanisms of glioma cell infiltration and invasion into the surrounding brain.

Suitability of the DNA content in urine stained material was evaluated for use in forensic analysis. High molecular weight (HMW) DNA (>20 kb, where 1kb equals 1000 base pairs) was obtained in only 9% (4 out of 44) of laboratory produced urine stains using an organic extraction method. Slot blot hybridization analysis with a human specific repeat probe (SLI332) showed that these stains contained DNA of human origin. A group of 12 urine stains from the original 44 that did not yield any DNA was subjected to Chelex resin extraction followed by PCR amplification for genotyping of the Human Leucocyte Antigen (HLA) DQA1 locus. Typing was successfull on 5 of the 12 samples. The concentrated sediment from the original liquid urine portion of the samples was similarly subjected to Chelex extraction and HLA DQA1 typing. Six out of ten samples were successfully typed. Blood specimens collected from the urine donors to serve as typing controls yielded identical genotypes in all cases. PCR based analysis of u...

In our efforts to understand the infrared spectral features of cells and tissues, and the spectral changes occurring between normal and disease states, we reported previously a detailed correlation between histochemical/immunohistochemical and spectral results. These results suggested an increase of nucleic acid spectral contributions in neoplastic, as compared to normal, tissue samples. In the present paper, these studies are extended to report the spectral features of DNA and RNA separately in these tissue samples. This was accomplished by selectively digesting either DNA or RNA from tissue sections, leaving behind the protein matrix with nuclear/cytoplasmic RNA or the protein matrix with nuclear DNA, respectively. These results demonstrate that the spectral changes between normal and neoplastic tissue are mostly due to an enhanced signature of DNA in neoplastic tissue. This enhancement is sufficiently large to suggest that it is most likely due to an increased detectability of DNA, rather than an increase in concentration. Index Headings: Infrared microspectroscopy; Human tissue; Liver; Cancer; Enzyme digestion.