Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Approaches to preclinical screening of antiangiogenic agents.

Angiogenesis, or new blood vessel growth, is essential for the growth, invasion, and metastasis of solid tumors. The inhibition of this process, or antiangiogenesis, is a promising new therapeutic anticancer strategy. Several antiangiogenic compounds are currently in preclinical or clinical development for the treatment of cancer. However, the challenge for the discovery and characterization of antiangiogenic targets remains in developing efficient in vitro or in vivo preclinical angiogenesis screening assays to assess and compare antiangiogenic activity. Several semiquantitative or quantitative angiogenesis assays exist, including in vitro endothelial cell systems and ex vivo or in vivo neovascularization models utilizing mouse, rat, or human tissues. We describe the more common and cost-effective angiogenesis assays currently in use, summarizing their unique advantages and disadvantages. Since angiogenesis inhibition is a novel therapeutic modality towards controlling solid tumors, antiangiogenic drug development underlines the importance in describing, standardizing, and developing quantitative screening assays for the next generation of antiangiogenic agents.

Pigmented renal clear cell carcinoma with melanocytic differentiation.

Renal cell carcinoma (RCC) is the most common malignant tumor of the adult kidney, and its incidence has been steadily rising. RCC consists of several subtypes, each of which has its own clinical features, and cytogenetic and molecular characteristics. Recognizing histologic patterns of RCC is important not only for correct diagnosis, but also for providing insight into the biological behavior of the tumor and subsequent appropriate medical care for the patient. Pigments other than hemosiderin has been observed in RCC, but none of them have been proved to be melanin. Melanotic tumors, either primary or metastatic, are rare in the kidney. We present an unusual case of melanin-pigmented clear cell RCC with melanocytic differentiation, an unusual variant that may lead to errors in diagnosis and treatment.

Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium.

PURPOSE: The NKX-3.1gene is an androgen regulated prostate specific homeobox gene that is believed to have a vital role in normal prostate development. In mice the homologue NKx3.1 is exclusively expressed in prostate epithelium. In humans NKX3.1 expression is also restricted to the prostate but to our knowledge the cellular location has not been described. Furthermore, since NKX3.1 maps to chromosomal band 8p21, a region with high loss of heterozygosity in prostate cancer, the gene has been proposed to have tumor suppressor function. In this study we demonstrate that in human prostates NKX3.1 is expressed exclusively in secretory epithelial cells and the level of NKX3.1 expression remains invariant in normal tissue and in tissue showing various grades of prostate cancer. In the 19 cases examined the DNA sequences of the NKX3.1 gene were identical and no mutation was detected. MATERIALS AND METHODS: Frozen tissue from patients who underwent radical prostatectomy was used for this study. For in situ hybridization experiments a 377 bp fragment corresponding to a portion of the 3' untranslated region of the NKX3.1 gene was amplified by polymerase chain reaction and cloned into the pCRII plasmid vector Invitrogen. Antisense or sense [33P] uridine triphosphate labeled RNA probes were generated with SP6 or T7 RNA polymerase and hybridized to the tissue sections. Slides were exposed to photographic emulsion and visualized on autoradiography. Laser capture microdissection was performed to procure pure populations of malignant epithelium. DNA was isolated by digesting samples in proteinase K buffer. Polymerase chain reaction and direct sequencing was performed using standard protocols. RESULTS: In vitro hybridization showed that NKX3.1 expression was restricted to secretory epithelial cells within benign prostate glands. No expression was detected in stroma or infiltrating lymphocytes. NKX3.1 was expressed in all grades of malignant epithelium in all 25 cases examined. Direct sequencing of the coding region of NKX3.1 revealed the wild-type sequence in all 18 microdissected cancers analyzed. CONCLUSIONS: Based on our studies we propose that NKX3.1 gene expression is restricted to benign and malignant secretory epithelium within the prostate but NKX3.1 does not appear to be a classic tumor suppressor gene responsible for prostate cancer initiation. These findings are consistent with the role of NKX3.1 in the development of normal prostate epithelium and maintenance of normal secretory function. Thus, NKX3.1 may represent a useful molecular marker for benign and malignant prostate epithelium.

Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < or = 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.

Immunohistologic responses within dermal metastatic melanoma lesions of patients treated with a synthetic peptide vaccine.

Three patients with dermal metastatic melanoma lesions responding to a synthetic peptide vaccine (g209-2M) derived from the sequence of gp100 melanoma-associated antigen, along with either IL-2 or granulocyte-monocyte colony-stimulating factor were studied to characterize the immunologic response occurring within and around the lesions during therapy. Standard immunocytochemical techniques were used to study the T-cell response (CD3, CD4, and CD8), the B-cell response (CD20), and the expression of class II major histocompatibility complex (HLA-DR) antigens. Between 40 and 65 days after the initiation of vaccine therapy (more than 3 weeks after the second dose of vaccine), the gross tumor size decreased and the tumors from all three patients showed substantial histologic regression associated with increased numbers of tumor-infiltrating lymphocytes and melanophages. The increased lesional tumor-infiltrating lymphocytes consisted of CD3+ T cells and very few CD20+ B cells. In two of the three patients, the T-cell infiltrate occurring during the initial tumor regression consisted predominantly of CD8+ cells. The number of perivascular T cells surrounding small vessels adjacent to melanoma lesions also increased during the time of peak histologic tumor regression. Also during the course of vaccine therapy, the expression of HLA-DR by vascular endothelial cells of the small vessels adjacent to lesions increased in all three patients, and elevated endothelial expression of HLA-DR was maintained in two of the three patients. These results show that patients with metastatic melanoma, who responded to melanoma vaccine therapy, had a predominantly CD8+ T-cell infiltrate associated with a loss of tumor cells. As the tumor cells diminished, they were replaced by heavily pigmented melanophages.

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay.

Endostatin has demonstrated potent antiangiogenic and antitumor activity in mouse models. We have investigated the ex vivo rat aortic ring assay and a human vein model to assess the biological activity of murine and human endostatin. Rat aortic rings were exposed to recombinant murine endostatin (Spodoptera frugipera; Calbiochem, San Diego, CA) or recombinant human endostatin (Pichia pastoris; EntreMed, Rockville, MD). After 5 days, murine endostatin (500 microgram/ml) demonstrated inhibition of microvessel outgrowth with dose-dependent effects (down to 16 microgram/ml). No significant inhibition was observed with human endostatin in the rat assay. Human endostatin at 250 and 500 microgram/ml inhibited outgrowths from human saphenous vein rings after a 14-day incubation. Electron microscopy assessed the formation of basal lamina, confirming that the microvessels were progenitors of patent vessels. Immunostaining for Factor VIII or CD34 demonstrated that the microvessel cells were endothelial. BrdU incorporation assays supported the presence of proliferating endothelial cells, correlating with neovascularization from the aortic wall. We conclude that the rat aortic ring assay confirms the antiangiogenic activity of murine but not human endostatin, suggesting that the model may have species specificity. However, the human form shows biological activity against human vascular tissue.

Microcystic adnexal carcinoma associated with primary immunodeficiency, recurrent diffuse herpes simplex virus infection, and cutaneous T-cell lymphoma.

Cutaneous microcystic adnexal carcinoma (MAC) is a rare and poorly understood tumor that predominantly occurs in the head and neck. MAC usually affects people in their fourth and fifth decades. Some patients have had a history of radiation. We present a case of MAC occurring in the left antecubital fossa of an 18-year-old white woman with an unusual immunodeficiency syndrome. The patient also developed a squamous cell carcinoma, a cutaneous T-cell malignancy, and a perigastric leiomyoma. A congenital infection of herpes simplex virus (HSV) persisted throughout her life. The association of HSV infection with MAC and squamous cell carcinoma and that of peripheral T-cell lymphoma with Epstein-Barr virus is discussed in relation to her immunodeficiency.

Loss of heterozygosity on chromosome 13 is associated with advanced stage prostate cancer.

PURPOSE: In order to investigate the possible involvement of a tumor suppressor gene(s) on chromosome 13 in prostatic neoplasms, we performed loss of heterozygosity (LOH) analysis on normal and tumor pairs from 36 prostate cancer patients. MATERIALS AND METHODS: Pure DNA was obtained from carcinoma cells and normal epithelium by tissue microdissection. The DNA had previously been analyzed for LOH on chromosomes 8 and 16. After an initial pilot experiment to determine the region(s) of significant LOH from 9 loci on chromosome 13q, 3 loci at and near the Rb1 locus (D13S153, D13S1319, and D13S1303) were chosen for further study. RESULTS: The overall rate of LOH on chromosome 13 was 27.3%. Four tumors exhibited LOH at all 3 loci. Two tumors exhibited LOH at D13S153 but not at the other, more telomeric loci; two additional tumors had loss at D13S1303 or D13S1319 but not D13S153. These data suggest that a tumor suppressor gene involved in prostate cancer may be located just telomeric to Rb1. Analysis of clinical and pathological data from carcinomas with and without loss shows that chromosome 13q LOH is correlated with advanced stage prostate cancer. CONCLUSIONS: Our LOH data suggests that there may be a tumor suppressor gene telomeric to Rb1 that is potentially involved in prostate cancer progression. Identification of this gene may be valuable in providing diagnostic and prognostic information for prostate cancer patients.

Polypoid dysplasia in Barrett's esophagus: a clinicopathologic, immunohistochemical, and molecular study of five cases.

Dysplasia in Barrett's esophagus (BE) is a precursor to adenocarcinoma and most commonly occurs as a flat, grossly undetectable lesion. Rarely, dysplasia in BE may grow as a polypoid lesion. Most BE-associated polypoid dysplastic lesions have been referred to as "adenomas" because of their histological similarity to a colonic adenoma. BE-associated polypoid dysplastic lesions have been less well characterized than the flat type. Therefore, our aim was to characterize the clinicopathologic and molecular features of five cases of BE-associated polypoid dysplasia and to review the literature on this entity. The cases were evaluated clinically, histologically, immunostained for MIB-1 and p53, and genotyped for loss of heterozygosity (LOH) at the adenomatous polyposis coli (APC) locus. Mucosal biopsy specimens of five BE patients without dysplasia, and five BE cases with high-grade flat dysplasia, were used as controls. The study patients were all male (average age, 71 years) who presented with symptoms of gastroesophageal reflux disease. Endoscopically, all five cases had a well-defined sessile or pedunculated polypoid lesion ranging from 0.4 to 1.5 cm in size in the mid (n = 1) or distal (n = 4) esophagus and were associated with specialized-type BE (four long segment, one short segment). Histologically, the polyps consisted of intestinalized epithelium with low- and high-grade dysplasia. All five cases contained adenocarcinoma (four within the polyp, one in adjacent BE). All polyps showed increased cell proliferation in the form of surface MiB-1 staining and showed positive p53 staining. Three of three (100%) informative cases showed LOH at the APC locus in the dysplastic epithelium and in areas of adenocarcinoma. All five flat dysplasia controls also showed surface MIB-1 staining and p53 positivity, and three of three informative controls showed LOH for APC. None of the nondysplastic BE controls showed any of these findings. Three patients were treated with esophagectomy and two with polypectomy. All were alive, without metastasis, from 2 months to 6 years later. A literature review of esophageal "adenomas" uncovered 12 cases. Four of these had no clinical or pathological information, two were, in fact, gastric heterotopic lesions, one was composed entirely of intestinal-type epithelium, and five were polypoid dysplastic lesions similar to the cases described here (three male, two female; mean age, 59 years). Four of these five cases were associated with adenocarcinoma in the polyp (two intramucosal, two submucosal). In summary, BE-associated polypoid dysplasia share similar clinical, pathological, and molecular features as flat dysplasia and are often associated with adenocarcinoma. Thus, we agree with other authors who recommend that the term adenoma, which usually carries a benign connotation, be abandoned in favor of a descriptive diagnostic term, such as "BE-associated polypoid dysplasia." BE patients with this lesion should be considered strong candidates for esophageal resection similar to lesions of this kind that occur in inflammatory bowel disease.

Chromosome 16 allelic loss analysis of a large set of microdissected prostate carcinomas.

PURPOSE: To perform loss of heterozygosity (LOH) analysis on chromosome 16 in 102 highly purified DNA samples isolated from one or more adenocarcinomas, prostatic intraepithelial neoplasia (PIN), and matched benign prostatic epithelium from 95 radical prostatectomy patients. MATERIALS AND METHODS: Specimens were procured by microdissection of frozen tissue samples, thus ensuring that highly select pure populations of cells were obtained for DNA extraction and LOH analysis. Multiple microsatellite markers were used to determine allelic loss on chromosome 16q. RESULTS: Overall loss on 16q was seen in 31% of the cancers, and occurred more frequently in high stage cancers than low stage cancers. In contrast, allelic loss in PIN failed to exceed 6% at any of the loci that were examined. CONCLUSIONS: These results suggest that inactivation of a putative tumor suppressor gene on 16q may be involved in the progression of some prostate cancers.

Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofibromatosis type 1.

We report on a girl with familial neurofibromatosis type 1 (Nf1) who at age 11 had multiple osteolytic lesions of the right mandible and the distal femoral and proximal tibial metaphyses bilaterally. No other skeletal component was affected, and no abnormalities were present on skeletal radiographs of her parents. Histologic examination of the mandibular and right femoral lesions revealed a "multinucleated giant cell process." Results of repeated routine laboratory and bone metabolic studies were within normal limits. The lack of reliable histologic criteria and the diagnostic problems in distinguishing among central giant cell granulomata, giant cell tumors of the jaw, cherubism, brown tumors resulting from (occult) hyperparathyroidism, Jaffe-Campanacci syndrome, McCune-Albright syndrome, Noonan-like/multiple giant cell lesion syndrome, and multiple nonossifying fibromas of bone are discussed.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor.

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

The histological spectrum of hemangiopericytoma: application of immunohistochemical analysis including proliferative markers to facilitate diagnosis and predict prognosis.

Hemangiopericytoma (HPC) is an uncommon vascular neoplasm thought to be derived from pericytes. Prediction of patient outcome is difficult based what is currently known about these tumors and histological parameters alone. We compiled 27 cases of HPC and evaluated the spectrum of histological features to investigate whether there was any correlation between histology, immunostaining, prognostic markers, and patient outcome. The following parameters were evaluated: vasculature, histological pattern (solid, myxoid, trabecular, alveolar), degree of cellular pleomorphism, necrosis, mitoses, and giant cell content. Immunohistochemistry was performed to determine the reactivity for CD 31, CD34, vimentin, actin, cytokeratin, S100, actin, and SMA. Proliferative rate was analyzed using antibodies to PCNA and MIB1. Patient's age ranged from 8 months to 75 years (mean, 35; median, 31). Twenty of 27 cases were located in the extremities. The tumors were grossly described as lobulated and well circumscribed (n=12) and nonencapsulated (n=15). By histology, the characteristic ramifying or staghorn vasculature pattern was seen in all cases. A solid histological pattern was mixed with an alveolar pattern in three cases, trabecular pattern in six cases, and myxoid pattern in two cases. Tumor cells were uniform, polygonal to spindle-shaped, often with vesicular nuclei. Tumor giant cells were present in 9 of 27 cases; necrosis, in 11 of 27. Mitoses ranged from 0 to 14 per 10 high-power fields (HPF). Cellular pleomorphism was 1+ in nine cases, 2+ in 12 cases, and 3+ in six cases. Immunohistochemistry showed reactivity for CD34 and vimentin in all cases. Actin was focally positive in one case, and SMA was focally positive in another. CD 31, cytokeratin, and S100 were uniformly nonreactive. Proliferative index measured by PCNA and MIBI ranged between less than 1% and 40% of tumor cells. Follow-up was available in 22 cases and ranged from 1 year to 15 years. Seven patients had metastases, and two recurred locally. Thirteen patients had no evidence of disease at last checkup. Parameters associated with recurrences or metastases include a trabecular pattern, the presence of necrosis, mitoses, vascular invasion, and cellular pleomorphism. Features associated with an aggressive biological behavior can be identified histologically. There was some, but not total, correlation between proliferative markers and tumor aggressiveness.

Familial osteoma of the cranial vault.

A case of solitary osteoma of the parietal skull is described in a mother and her 12-year-old son. There were no other craniofacial or dysmorphic features and there was no history of previous cranial trauma or inflammatory process and no radiographic evidence of other skeletal lesions in both patients. Family history was otherwise unremarkable. Gardner syndrome was ruled out by excluding other associated clinical abnormalities and by family history. The clinical and radiological features of the patients' osteomas were different from those of Albright hereditary osteodystrophy, fibrodysplasia ossificans progressiva, progressive osseous heteroplasia and familial ectopic ossification. To the author's knowledge this is the first case of the familial presentation of osteomas.

Allelic deletion at chromosome 9p21(p16) and 17p13(p53) in microdissected sporadic dysplastic nevus.

A critical area of chromosomal loss at region p16(9p21-22) and p53(17p13) has been implicated in the genesis of malignant melanoma. It is still unclear whether the genetic alterations can be detected in dysplastic nevus, a premalignant lesion of malignant melanoma. We have searched the frequency of p16 and p53 deletion in nine dysplastic nevi and 13 benign intradermal nevi with five microsatellite markers. Hemizygous deletion was detected in seven of nine (78%) dysplastic nevi at one or more loci for p16 and three of seven (43%) for p53, respectively. No loss of heterozygosity (LOH) was detected in any of the benign intradermal nevi. All three dysplastic nevi with LOH for p53 also showed LOH at p16. However, not all dysplastic nevi showing p16 deletion showed p53 gene deletion. Therefore, these data suggest that deletion of p16 may play an important role in the development of dysplastic nevus as an early event and that the changes may represent an early event in the development of malignant melanoma.