Expression of telomerase subunits in normal and neoplastic prostate epithelial cells isolated by laser capture microdissection.

BACKGROUND: Vertebrates have special structures at the ends of their chromosomes, known as telomeres, which may provide the chromosomes with stability and protect them from exonucleolytic degradation. The shortening of telomeric DNA with each cell division may lead to cell cycle arrest and/or apoptosis of a normal human somatic cell. Telomerase, an RNA-dependent DNA polymerase, elongates the 3'-ends of telomeric DNA. Thus, the presence of telomerase activity may reflect a cell's potential immortal state. The telomerase complex is comprised of several subunits. In the current study, the authors describe the use of laser capture microdissection (LCM) to procure pure matched tumor and normal cell populations from histologic sections and to determine the expression of telomerase subunits in these purified samples. METHODS: Pure matched tumor and normal prostate epithelial cells were procured by LCM using fresh frozen tissue samples obtained from patients undergoing radical prostatectomy. RNA was extracted from LCM captured cells, and the subunits for telomerase were assayed by reverse transcriptase-polymerase chain reaction. RESULTS: In 18 samples that were captured with LCM, only the catalytic subunit of telomerase, or hTERT, was found to be discriminatory between tumor cells (17 of 18 specimens, 94.4%) and nontumor cells (none of 18 specimens). TP1, a protein that has been shown to be associated with telomerase activity, was expressed in 3 of 18 normal cells (16.7%) and 15 of 18 tumor cells (83.3%). The RNA subunit of telomerase, or hTR, was expressed in 10 of 18 normal cells (55.6%) and 18 of 18 tumor cells (100%). There was no apparent correlation between telomerase subunit(s) expression and Gleason sum score. CONCLUSIONS: Molecular analyses of LCM cells from prostate carcinoma patient samples demonstrated transcriptional up-regulation of all telomerase subunits in the prostatic epithelium. However, only the catalytic subunit of telomerase, hTERT, was found to be discriminatory between neoplastic versus normal cells (94.4% vs. 0%). This finding suggests that the hTERT subunit may be a useful marker for the detection of prostate carcinoma and/or a potential target for therapy.

Genetics of epithelial tumors of the renal parenchyma in adults and renal cell carcinoma in children.

The classification of renal cell carcinomas has been affected by both the delineation of new histologic subtypes and the understanding that recognized histomorphologic patterns are reflective of specific sets of cytogenetic abnormalities. In fact, standard methods of clinicopathologic study and cytogenetic analysis have been cooperatively contributory in the evolution of current concepts regarding the biologic nature and classification of renal parenchymal epithelial tumors. In this review, the current classification scheme for these tumors is discussed from the perspective of both the defining histologic and cytogenetic features. Limited molecular data are described as well.

An animal model for delayed hemolytic transfusion reactions.

Delayed hemolytic transfusion reactions (DHTRs) are a well-known complication of transfusion that may be defined as immune-mediated hemolysis of allogeneic donor red cells that occurs approximately 3 to 5 days after transfusion. In general, DHTRs occur in patients who have been alloimmunized previously, but the antibody titers have fallen below serologically detectable levels. Transfusion of seemingly compatible blood and exposure to the putative alloantigen results in an anamnestic immune response that may lead to in vivo accelerated destruction of donor red cells. Symptoms may include a drop in hemoglobin and hematocrit, fever, jaundice, and renal insufficiency. More recent studies have shown that there is a subset of cases called delayed serologic transfusion reactions (DSTRs) when there are serologic findings consistent with DHTRs but no clinical evidence of hemolysis. In both DHTRs and DSTRs, direct antiglobulin tests are often persistently positive long after the transfused donor red cells should have been removed from the circulation. Because the studies required to investigate the immunologic and clinical aspects of these reactions are precluded in humans, we developed an animal model for the study of DHTRs and DSTRs. Our article provides a comprehensive review of DHTRs and DSTRs, the role of complement and cytokines in these reactions, and the phenomenon of bystander hemolysis. We describe our studies using the rabbit as a model for the study of DHTRs and bystander hemolysis.

Dermatomyositis as a presentation of pulmonary inflammatory pseudotumor (Myofibroblastic tumor).

Inflammatory pseudotumor (IPT) is a rare pulmonary tumor of uncertain etiology that usually presents as an asymptomatic radiographic finding. We describe a case of pulmonary IPT presenting as dermatomyositis with complete resolution following surgical resection.

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.

Mutually exclusive expression patterns of Bcl-2 and Par-4 in human prostate tumors consistent with down-regulation of Bcl-2 by Par-4.

Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis. Constitutive- or regulated- overexpression of Par-4 caused a reduction in the levels of the anti-apoptotic protein Bcl-2. Replenishment of Bcl-2 levels abrogated susceptibility to Par-4-dependent apoptosis, suggesting that Par-4-mediated apoptosis requires downmodulation of Bcl-2 levels. The inverse correlation between Par-4 and Bcl-2 expression was recapitulated in human prostate tumors. Par-4 but not Bcl-2 was detected in the secretory epithelium of benign prostatic tumors and in primary and metastatic prostate cancers that are apt to undergo apoptosis. Moreover, xenografts of human, androgen-dependent CWR22 tumors showed Par-4 but not Bcl-2 expression. By contrast, androgen-independent CWR22R tumors derived from the CWR22 xenografts showed mutually exclusive expression patterns of Par-4 and Bcl-2. These findings suggest a mechanism by which Par-4 may sensitize prostate tumor cells to apoptosis.

Digital image analysis of proliferative index: two distinct populations of high-grade prostatic intraepithelial neoplasia in close proximity to adenocarcinoma of the prostate.

A considerable amount of data has been collected showing the association of high-grade prostatic intraepithelial neoplasia (HGPIN) with adenocarcinoma of the prostate, and many studies have yielded results that suggest that HGPIN is a precursor of carcinoma. A few studies have indicated that HGPIN may, in some cases, be a sequela of prostatic adenocarcinoma. We examined the proliferative indices of HGPIN, carcinoma, and benign prostatic epithelium by computer-aided counting of Ki-67-positive nuclei in 15 cases in which HGPIN and carcinoma were in close proximity. There were 13 radical prostatectomy specimens with prostate cancer and two cystoprostatectomy specimens with both transitional cell carcinoma and prostatic adenocarcinoma. First, we showed the accuracy of the computer-aided counting method compared with direct counting through the binoculars of the microscope. Then proliferative activity was assessed for each case by picking the two areas of carcinoma, the two areas of HGPIN, and the one area of benign epithelium with the greatest density of carcinomatous, dysplastic, and benign Ki-67-positive nuclei, respectively. The total number of nuclei and the number of positive nuclei were counted. Basal cells were not counted. The mean proliferative index was higher for cancer (caindex, average 0.054) than for HGPIN (pinindex, average 0.048) (P < .05). We found that the 15 cases fell into two distinct groups. The average ratio of pinindex to caindex (pinindex/caindex) was lower in group 1 (0.72) than in group 2 (1.54) (P=.17), and when the results were corrected for the nonzero gamma-intercepts of the regression lines of pinindex versus caindex, the ranges were widely separated, and the difference between the means was statistically significant (0.15 v 0.62; P < .0001). A greater subjective similarity between the nuclear features in the HGPIN and those of the corresponding carcinoma was noted for the cases in group 2. The average value of bngnindx was 0.014. The value of bngnindx did not correlate with either caindex or pinindex. We conclude that there may be two types of lesions with the morphological appearance of HGPIN and that they may have different relationships to carcinoma. Computer-aided counting of digitized microscopic images is both labor-saving and as accurate as enumeration directly through the binoculars of the microscope.

Diagnoses rendered on prostate needle biopsy in community hospitals.

BACKGROUND: Reported incidences of various diagnoses made on needle biopsy of the prostate vary significantly in the literature, most of which has originated from large, academic medical centers. METHODS: We recorded all the prostate needle biopsy results from three community hospitals for 1990-1993 to determine the rates of, and trends in, various diagnoses in these practices. RESULTS: Hospital H1 (1,192 cases) halved the rate of atypical, nondefinitive diagnoses from 11.8% in 1990 to 5.7% in 1993 (P < 0.001). The rate at H2 (2,792 cases) remained essentially unchanged at 5.95+/-0.55%, and H3 (1,306 cases) went from 2.3% to 6.0% (0.1 < P < 0.2). In the setting of an atypical, nondefinitive diagnosis, H1 and H2 recommended repeat biopsy less than 7% of the time. H3 made this recommendation in an average of 22.1% of atypical cases. Annual rates of high-grade prostatic intraepithelial neoplasia (PIN) showed no trend over time, and averaged 2.0% (1.2-3.25%) at H1 and 1.2% (0.3-2.0%) at H2. The diagnosis was never made at H3. The fraction of cancers diagnosed as low-grade (Gleason sum < or = 4) showed a statistically significant decreasing trend over time at all three hospitals (P < 0.05). These data are compared with those from the Johns Hopkins Hospital (JHH), a large academic center in geographic proximity to hospitals H1-H3. CONCLUSIONS: At these three community hospitals, we discerned (1) convergence to a rate of approximately 6.0% of atypical, nondefinitive diagnoses; and (2) a progressively more appropriate fraction of carcinomas diagnosed as low-grade on needle biopsy. The rates of diagnosis of high-grade PIN and recommendation of repeat biopsy varied. These rates of PIN, atypical, nondefinitive diagnoses, and low-grade cancer represent an assessment of diagnostic habits.

Immunohistochemical analysis of the proapoptotic protein Par-4 in normal rat tissues.

Prostate apoptosis response 4 (par-4) is a recently identified gene that encodes a transcription factor, Par-4, with a leucine zipper domain. Par-4 protein is constitutively expressed in various cell lines and is functionally required but not sufficient for apoptosis. Induction of Par-4 in cultured cells is found exclusively during apoptosis, and ectopic overexpression of Par-4 enhances the potency of apoptotic stimuli. Western or Northern blot analysis on mRNA or protein extracts, respectively, from rat organs revealed that the expression of Par-4 was ubiquitous and was not restricted to any specific organ(s). To further identify specific cell types that expressed Par-4, we performed an immunohistochemical analysis of the protein in paraffin-embedded sections of various organs from rats. Our findings indicated that consistent with its proapoptotic role, Par-4 is expressed in apoptotic granulosa cells of atretic ovarian follicles and in terminally differentiated cells, such as the cardiomyocytes, cerebellar Purkinje cells, and pyramidal cells of the hypothalamus. Moreover, testosterone ablation by castration of rats caused an early and transient induction of Par-4 in the ductal cells of the prostate that undergo apoptosis. By contrast, in tissues in which the cells could be visually differentiated from their mature counterparts, Par-4 expression was lowest in the mature cells. This was the case for epithelia of the mammary and the prostate gland in which the basal cells maintained higher protein levels of Par-4 than did the terminally differentiated ductal cells. Similarly, cells of the stratum corneum of the skin and cells on top of the duodenal villi stained less intensely for Par-4 as compared to the stem cells in the stratum basale and at the bottom of the crypts of Lieberkühn, respectively. It is possible that Par-4 has to be down-regulated for successful differentiation in these tissues. Taken together, the widespread expression of Par-4 in various adult cell types underscores the physiological importance of the protein. The observation of constitutive Par-4 expression in the stem cell compartments is inconsistent with the probability of apoptosis per se and can be extended to determine whether Par-4 plays a role in other cellular processes.

Telepathology diagnosis of prostrate needle biopsies.

We conducted a prospective analysis of the diagnostic accuracy of a static-image telepathology system (Roche RIAS, Elon College, NC) in the interpretation of needle biopsies of the prostate (NBx). Two hundred consecutive cases received in consultation were included. Each case was examined by one of the researchers (MHW), and images were captured either according to the areas of concern designated by the referring pathologist (set A; 100 cases) or according to the judgment of MHW (set B; 100 cases). The other researcher (JIE) daily rendered diagnoses first on the video images and then by direct microscopy. Accuracy of video diagnosis was categorized as 0 (correct), 1 (minor error), 2 (major error), or 3 (deferred). An average of 5.49 images were captured per case in set A, and 5.28 for set B. Seventy-seven, 9, 9, and 5 cases were categorized as 0, 1, 2, and 3, respectively, for set A, and 78, 17, 1, and 4 cases, respectively, for set B. Video versus direct diagnoses for the type 2 errors were five carcinoma versus markedly atypical, two carcinoma versus atypical, one carcinoma versus nonspecific granulomatous prostatitis, and two benign versus atypical. In these difficult NBx, telepathology allowed an essentially correct diagnosis in almost all of the cases. The number of images required was reasonable, and the images were of excellent quality. However, the accuracy varied from set A to set B, with the fractions of nondeferred cases that were given an essentially correct video diagnosis totaling 91% and 99%, respectively (P < .01). Accuracy of telepathology diagnosis using static images may depend on the person capturing the images, even in the case of small biopsies.

Neuroendocrine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy.

It is controversial whether neuroendocrine (NE) differentiation in adenocarcinoma of the prostate is associated with more aggressive behavior. Most studies included patients with tumors of a wide range of grades and stages and an end point of disease-specific survival, a relatively insensitive marker of progression. The authors studied completely embedded radical prostatectomy specimens from 104 patients with clinically organ-confined carcinoma and no history of adjuvant or neoadjuvant therapy. Progression was marked by a serum prostate-specific antigen (PSA) concentration greater than or equal to 0.2 ng/mL. Seventy-six men did not progress, with a mean follow-up period of 8.0 years (range = 7 to 10 years). Forty-eight men progressed at a mean time after surgery of 3.6 years (range = 1 to 8 years). Twenty-one percent of the tumors were organ confined: 79% had capsular penetration. Seminal vesicles and lymph nodes were negative in all cases. A representative section through the main tumor mass was stained for chromogranin A. Reactive neoplastic cells were counted subjectively as well as individually enumerated. Gleason grade, pathological stage, and degree of NE differentiation all correlated with progression. Only grade and extent of NE differentiation predicted progression in a multivariate analysis. NE differentiation did not correlate with stage or grade. Extent of NE differentiation separated patients (59 cases) with tumors of Gleason sum less than or equal to 6 into groups with high and low risks for progression (P < .008) independent of Gleason sum. Extent of NE differentiation provides prognostic information in addition to that provided by grade in cases of early prostate cancer treated by radical prostatectomy.

Significance of high-grade prostatic intraepithelial neoplasia on needle biopsy.

We studied 33 cases with an initial needle biopsy of the prostate that showed only high-grade prostatic intraepithelial neoplasia (PIN 2-3), for which follow-up biopsies were available. Twenty-four men (73%) were shown to have adenocarcinoma either on a simultaneous (14 patients) or subsequent (10 patients) biopsy. The grade of PIN (grade 2 v 3), rectal examination findings, and transrectal ultrasound results proved not to be significantly different in patients with proven adenocarcinoma compared with those without proven carcinoma. In contrast, serum prostate-specific antigen (PSA) concentrations were elevated in 90% of patients with carcinoma compared with only 50% of those with a benign follow-up biopsy. Persistent elevation of serum PSA concentration was seen in only one of three patients with serial PSA measurements and a benign follow-up biopsy. Notably, all patients with carcinoma for whom we had serial measurements of serum PSA levels had persistent elevation. The finding of high-grade PIN on needle biopsy often represents a sampling problem with carcinoma nearby. Consequently, the finding of high-grade PIN on needle biopsy merits vigorous follow-up, including rebiopsy. In particular, patients with increased serum PSA appear to be at greater risk of harboring prostatic adenocarcinoma. However, a significant number of patients with high-grade PIN on initial biopsy may not have evidence of carcinoma on repeat biopsy. Thus, radical prostatectomy or radiotherapy for PIN is not warranted.

Temporary external shunt bypass in the traumatically amputated upper extremity.

The technique of temporary external shunt bypass offers early perfusion of a traumatically amputated extremity, thus decreasing the total anoxic time, permitting bony fixation after perfusion is established, and allowing arterial revascularization with good venous outflow and minimal blood loss. This technique may increase the survival of these severely damaged limbs.

Sexual function after aorto-lliac surgery.

Patients undergoing aorto-iliac operations frequently suffer severe physical and psychological problems as a consequence of postoperative sexual disability. In an attempt to reduce this symptom complex, careful preoperative and postoperative evaluations were performed on 20 patients undergoing such procedures. Their average age was 64 years. Patients were divided into groups distinguished by type of operative procedure and extent of sexual function, then carefully evaluated for changes related to technical aspects of the vascular reconstruction. Data were accumulated pertaining to preoperative and postoperative frequency of sexual intercouse, ability to achieve and maintain an erection, occurrence of retrograde ejaculation, and other symptoms of genitourinary dysfunction. Significant and disabling sexual dysfunction, found to occur after routine abdominal aneurysmectomy, aorto-femoral bypass grafting, and aorto-iliac endarterectomy, is believed to be related to type of operation and technical aspects of the procedure. The incidence of postoperative retrograde ejaculation and impotence was significant.