Genetic screening for von Hippel-Lindau gene mutations in non-syndromic pheochromocytoma: low prevalence and false-positives or misdiagnosis indicate a need for caution.

Genetic testing of tumor susceptibility genes is now recommended in most patients with pheochromocytoma or paraganglioma (PPGL), even in the absence of a syndromic presentation. Once a mutation is diagnosed there is rarely follow-up validation to assess the possibility of misdiagnosis. This study prospectively examined the prevalence of von Hippel-Lindau (VHL) gene mutations among 182 patients with non-syndromic PPGLs. Follow-up in positive cases included comparisons of biochemical and tumor gene expression data in 64 established VHL patients, with confirmatory genetic testing in cases with an atypical presentation. VHL mutations were detected by certified laboratory testing in 3 of the 182 patients with non-syndromic PPGLs. Two of the 3 had an unusual presentation of diffuse peritoneal metastases and substantial increases in plasma metanephrine, the metabolite of epinephrine. Tumor gene expression profiles in these 2 patients also differed markedly from those associated with established VHL syndrome. One patient was diagnosed with a partial deletion by Southern blot analysis and the other with a splice site mutation. Quantitative polymerase chain reaction, multiplex ligation-dependent probe amplification, and comparative genomic hybridization failed to confirm the partial deletion indicated by certified laboratory testing. Analysis of tumor DNA in the other patient with a splice site alteration indicated no loss of heterozygosity or second hit point mutation. In conclusion, VHL germline mutations represent a minor cause of non-syndromic PPGLs and misdiagnoses can occur. Caution should therefore be exercised in interpreting positive genetic test results as the cause of disease in patients with non-syndromic PPGLs.

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

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.

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.

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.

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.

Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors.

OBJECTIVE: Our objective was to determine the frequency of allelic loss at 8p21 in sporadic epithelial ovarian cancer. We recently described allelic loss at this locus in 7/9 ovarian cancers from patients with BRCA1 gene mutations. METHODS: We anonymously obtained and examined 40 unselected invasive epithelial ovarian cancers and 5 low-malignant-potential (LMP) ovarian tumors for loss of heterozygosity (LOH) at 8p12-22. Pure epithelial and stromal cell populations were procured selectively by laser capture microdissection and extracted DNA was amplified with polymorphic microsatellite markers spanning the region of interest. RESULTS: LOH was highest (50%) at marker D8S136 located at 8p21 with 15 of 30 informative cases exhibiting an allelic deletion. None of the LMP tumors evaluated showed LOH at 8p12-22. A trend toward more frequent LOH at 8p12-22 was identified with increasing disease aggressiveness from LMP to early stage invasive ovarian cancer to advanced stage invasive ovarian cancer (Lehman's test, P2 < 0.024). CONCLUSIONS: Fifty percent allelic loss at the distal portion of 8p21 has not been reported to date for sporadic epithelial ovarian carcinomas. The higher rate of loss in our cohort, in contrast to previous allelotyping studies, is due likely to analysis from homogenous cell populations. These results, in concert with our previous study of BRCA1 mutation-positive patients, suggest a tumor suppressor gene locus at 8p21 for epithelial ovarian cancer.

Hypercalcemia in patients with metastatic renal cell carcinoma: effect of nephrectomy and metabolic evaluation.

PURPOSE: The role of nephrectomy in the management of hypercalcemia in metastatic renal carcinoma is not known. Hypercalcemia in patients with renal cell carcinoma frequently mimics primary hyperparathyroidism and has been attributed to tumor secretion of parathyroid hormone related protein. We determined the role of cytoreductive surgery in patients with metastatic renal cell carcinoma and hypercalcemia, identified factors that predict patient benefit from surgery, and evaluated the mechanisms of hypercalcemia in these patients. MATERIALS AND METHODS: A total of 15 patients with metastatic renal cell carcinoma and hypercalcemia underwent metabolic and laboratory evaluation followed by nephrectomy. Post-operatively they were followed for changes in serum calcium levels. We selected 18 normocalcemic patients with metastatic renal cell carcinoma and 4 normocalcemic patients without renal cancer to serve as control groups for survival and parathyroid hormone related protein expression. RESULTS: A decrease in serum calcium corrected for albumin occurred in 9 of 11 patients at 1 to 4 weeks after nephrectomy and in 7 of 12 patients at 5 to 16 weeks after nephrectomy. Clinical evaluation supported a parathyroid hormone related protein mechanism of hypercalcemia in 5 of 8 patients. Two patients had evidence of local osteolytic hypercalcemia and 1 had prostaglandin mediated hypercalcemia. CONCLUSIONS: Nephrectomy temporarily ameliorated hypercalcemia in a subgroup of patients with metastatic renal cancer and hypercalcemia. Parathyroid hormone related protein expression was commonly found to be associated with hypercalcemia. Nonparathyroid hormone related protein mechanisms of hypercalcemia in renal carcinoma may be more common than previously thought.

Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma.

OBJECTIVES: To identify differentially expressed genes in tumor cells of patients with prostate cancer by means of tissue microdissection and targeted differential display. METHODS: RNA was recovered from pure populations of microdissected normal epithelium and invasive tumor from frozen tissue sections of a radical prostatectomy specimen. Reverse transcription-polymerase chain reaction (PCR) using arbitrary and zinc finger PCR primers was performed. RESULTS: A 130-base pair product was identified that appeared selectively in the tumor sample. DNA sequence analysis revealed it to be a clone from the expressed sequence tag database (GenBank accession R00504). Microdissection of normal epithelium and the corresponding invasive tumor was subsequently performed on a test panel of 10 prostate carcinoma specimens. Comparison of R00504 levels in normal epithelium and invasive carcinoma, using beta-actin as an internal control, showed the transcript to be substantially overexpressed in 5 of 10 carcinomas. Northern blotting revealed R00504 to be a 2.6-kilobase gene. CONCLUSIONS: A novel transcript up-regulated in an aggressive prostate carcinoma was identified using degenerate zinc finger primers in microdissected tissue samples. The approach used in this study may be helpful in quantitative comparison of known genes and identification of novel genes in microdissected human tissue samples.

Generation and genetic characterization of immortal human prostate epithelial cell lines derived from primary cancer specimens.

Difficulty in establishing long-term human prostate epithelial cell lines has impeded efforts to understand prostate tumorigenesis and to develop alternative therapies for prostate cancer. In the current study, we describe a method that was successful in generating 14 immortal benign or malignant prostate epithelial cell cultures from primary adenocarcinomas of the prostate resected from six successive patients. Immortalization with the E6 and E7 transforming proteins of human papilloma virus serotype 16 was necessary to establish long-term cultures. Microscopic examination of fresh tumor specimens exhibited a variable mixture of benign and malignant epithelium. Thus, single-cell cloning of tumor-derived cell cultures was essential for defining tumor cell lines. Efforts to characterize these cultures using traditional criteria such as karyotype, growth in nude mice, and prostate-specific antigen expression were noninformative. However, allelic loss of heterozygosity (LOH) represents a powerful alternative method for characterizing tumor cell lines originating from primary adenocarcinomas of the prostate. Microdissected fresh tumors from four of six patients revealed LOH at multiple loci on chromosome 8p, as assessed by PCR. LOH on chromosome 8p matching the patterns found in microdissected tumors was also observed in a tumor-derived cell line and its clones, as well as in one clone from a tumor-derived cell line from a second patient. LOH was not observed in immortal lines generated from autologous benign prostatic epithelium, seminal vesicle epithelium, or fibroblasts. The multifocal nature of prostate cancer, as well as the presence of an entire spectrum of malignant transformation within individual prostate glands, necessitates this type of careful analysis of derivative cell cultures for their validation as in vitro models that accurately reflect the primary cancers from which they are derived.

Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21.

To investigate the possible involvement of a tumor suppressor gene(s) on chromosome 8 in prostatic neoplasms, we performed a comprehensive loss of heterozygosity (LOH) study on 99 tumors from 97 prostate cancer patients. One of the carcinomas was a lymph node metastasis; the other 98 were primary carcinomas. Pure populations of carcinoma cells and normal epithelia were procured by tissue microdissection. Two separate tumor foci were obtained from each of two patients. Microsatellite markers from 25 loci on the short arm and one locus on the long arm of chromosome 8 were used for PCR-based LOH analysis on matched normal and tumor DNA samples. The overall LOH on 8p in this study was 85.9% (85 of 99) of carcinomas. The loss was highest at markers D8S133, D8S136, NEFL, and D8S137 (62,72, 64, and 75%, respectively), which are located at 8p12-21. Seventy-nine of 99 tumors exhibited loss in at least one of these four loci. In contrast, LOH at 8p22 was much lower: 17,18,18, and 19% at D8S549, D8S602, D8S254, and D8S261, respectively, with 25 of 99 tumors showing deletion in one or more of the four loci. All but 5 tumors with deletions in this more distal region had at least one retained locus between the 8p22 deletion and a more proximal region of loss at 8p12-21; 1 tumor had loss at 8p22 but not 8p12-21. This suggests there may be two distinct regions of loss and, therefore, two tumor suppressor genes on this chromosomal arm. The loss on 8p12-21 showed little or no correlation with grade or stage of disease.

Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia.

The development and progression of human prostate cancer is associated with genetic abnormalities in tumor cells. Inactivation of tumor suppressor genes due to allelic loss is thought to be an important mechanism of gene alteration in prostatic neoplasms. In this study we examined allelic loss on chromosome 8p12-21 in microdissected samples of normal prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and invasive prostate carcinoma from the same patients. Tissue microdissection under direct microscopic visualization procures pure populations of cells of interest, including small lesions such as PIN. Among 30 patients with concomitant cancer and PIN, we found loss of heterozygosity on chromosome 8p12-21 in 63% (34 of 54) of foci of PIN examined and 90.6% (29 of 32) of tumors, suggesting that abnormalities on chromosome 8p12-21 may be important in the early stages of prostatic carcinoma development. Several cases in which multiple foci of PIN from the same patient were sampled showed different patterns of allelic loss. Fifty-five % (16 of 29) of the prostate carcinomas contained a potential precursor PIN focus based on allelic loss pattern. Our results are consistent with the hypothesis that PIN arises multifocally within the prostate gland, and that a subset of these lesions progress to become carcinoma.

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.

Electrophoretic karyotype for Dictyostelium discoideum.

This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.

Analysis of developmentally expressed antigens in Polysphondylium pallidum.

A series of monoclonal antibodies were previously raised against developing Polysphondylium pallidum cells. In this work, six of these antibodies have been used as probes to identify and characterize antigens regulated during development. Soluble and membrane fractions of P. pallidum cells at six stages of development or three stages of cyclic adenosine monophosphate (cAMP)-induced development were run in sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analysis. Three of the monoclonals, anti-Tp200, anti-Tp423, and anti-Pg101, stain sorogen tips. Tp423 and Tp200 are membrane-associated antigens; both are stable to urea extraction, and Tp200 remains in the membrane after NaOH extraction. Tp423 is present in starved cells but is more prominent in sorogens and particularly in cAMP-developed cells. In contrast, Tp200 is first detected in early to mid-aggregation and is more abundant late in development. Pg101, which is expressed as a gradient with its highest concentration in tips, first appears in tight aggregates but is much more abundant in sorogens; unlike the Tp antigens, Pg101 is not greatly induced in cAMP-developed cells. All three of these antigens undergo changes in apparent molecular weight at the tight aggregate or sorogen stage: The gel mobilities of Tp200 and Pg101 increase, whereas that of Tp423 decreases. In addition to the tip-specific monoclonals, two monoclonals that stain all but the tips of sorogens have been used for analysis. One of these, anti-3D10Pnk stains most cells within secondary tips, whereas anti-3D10Dif does not. 3D10Dif is membrane associated; it is present very early in development, increasing two- to threefold through the sorogen stage and diminishing in late cAMP-developed cells. 3D10Pnk is a mostly soluble species first detected in late streaming. Anti-1c3, a sixth monoclonal, which stains nuclei uniformly throughout sorogens, is also developmentally expressed. 1c3 is mainly membrane associated and is expressed from late streaming through the sorogen stage.