Analysis of gene amplification in archival tissue by differential polymerase chain reaction.

Oncogene amplification is found in many human tumors, and its detection may have important prognostic value. However, analysis of gene amplification may be hampered by inadequate tissue or poor DNA quality. We have previously described a polymerase chain reaction (PCR)-based procedure called differential PCR that can detect variations in gene dosage using miniscule amounts of tumor DNA [Frye, R.A., Benz, C.C. & Liu, E. (1989). Oncogene, 4, 1153-1157]. We now report the optimization of this technique for the analysis of oncogene amplification in paraffin-embedded archival tissues. We find that differential PCR is able to detect amplification of the HER2 (c-erbB-2) and the epidermal growth factor receptor (EGFR) genes and can be used to arrive at a semiquantitative estimate of gene dosage. Furthermore, our approach can determine gene amplification in samples in which the DNA is significantly degraded. Using differential PCR on paraffin-embedded tissues from cases previously investigated by standard DNA extraction and dot-blot procedures, good correlation between the two methods was found. Approaches are described to overcome technical problems posed by factors that affect the differential PCR, including the method of DNA extraction and extreme fragmentation of the DNA (less than 200 base pairs). Furthermore, the resulting analytical algorithm reported herein has proved effective in detecting oncogene amplification in archival breast cancer specimens from standard pathology laboratories. Thus, differential PCR will be particularly helpful in the analysis of tumor specimens that are archived, small in size or rare in occurrence.

FDG PET for detection and therapy control of metastatic germ cell tumor.

UNLABELLED: We investigated the use of PET and 2[18F]fluoro-2-deoxy-D-glucose (FDG) for detection and therapy control of metastatic germ cell cancer in comparison to CT. METHODS: Fifty-four PET studies were performed in addition to CT in 33 patients with histopathologically proven germ cell tumors (14 seminomas, 18 nonseminomas, 1 not classified). The scans were done either after initial diagnosis (Group 1; n = 12), within 2 wk after completion of chemotherapy (Group 2; n = 13) or 14-375 days after chemotherapy (Group 3; n = 29). PET and CT were validated either by histology (n = 19) or clinical follow-up for 182-1704 days (n = 35). Focal pathological uptake with PET was quantified using standardized uptake values (SUVs). RESULTS: PET was significantly more accurate than CT (0.86 versus 0.59; p < 0.025) for detection of residual viable tumor in Group 3. While sensitivities of PET and CT did not differ markedly, PET was significantly more specific than CT. No significant differences between PET and CT were found in Groups 1 and 2. PET scans after therapy resulted in false-negative findings in five of nine cases of Group 2 but only in two of nine cases of Group 3. False-positive PET findings occurred in three inflammatory processes. SUV of seminomas was significantly higher than in nonseminomas (p < 0.01). CONCLUSION: PET using FDG is superior to CT for assessment of residual tumor after chemotherapy of germ cell cancer and may thus have an increased effect on patient management in the future. PET must be performed at least 2 wk after completion of therapy. Further data are necessary to determine the role of FDG PET for initial staging of germ cell cancer.

Cytogenetic analysis of 11 renal oncocytomas: further evidence of structural rearrangements of 11q13 as a characteristic chromosomal anomaly.

We carried out cytogenetic analysis on 11 renal oncotytomas by using G-banding and DAPI-banding techniques. Four of our tumors exhibited structural rearrangements affecting chromosome 11 at band q13. Together with another case previously described by us, our tumors constitute the largest series of renal oncocytomas displaying translocations involving 11q13. A review of the literature disclosed only 6 similar oncocytomas, 1 tumor with a t(9;11)(p23;q12), 2 tumors with a nearly identical t(9;11)(p23;q13), and 3 tumors with a t(5;11)(q35;q13). Therefore, our findings provide further cytogenetic evidence that genes located on 11q12-13 may be involved in the tumorigenesis of renal oncocytomas.

Expression of glucose transporter 1 (Glut-1) in cell lines and clinical specimens from human prostate adenocarcinoma.

BACKGROUND: Increased uptake and metabolism of glucose is a characteristic of malignant transformation. Overexpression of glucose transporters, especially Glut-1, is a common event in human malignancies. To date, little is known about the role of Glut-1 in human prostate cancer (PC). The aim of this study was to investigate the expression of Glut-1 both in PC cell lines and clinical specimens of primary PC. MATERIALS AND METHODS: The PC cell lines DU145, PC3 and LNCaP were assessed for Glut-1 mRNA expression by Northern blot analysis. In a total of 45 primary PC specimens, radioactive (35S) in situ hybridizations (RISH) for Glut-1 mRNA expression were performed on frozen sections. Quantification of Glut-1 expression was obtained by use of an image analysis system. RESULTS: Glut-1 expression was detected in all 3 cell lines. Expression in the more poorly-differentiated cell lines DU145 and PC3 was even higher than in the hormone-responsive LNCaP cell line. In situ hybridizations in primary PC revealed Glut-1 expression just above the detection limit in well-differentiated tumors. Significantly increased Glut-1 expression was detected in moderately- to poorly-differentiated PC. CONCLUSION: Glut-1 is expressed in PC cell lines and primary PC. The level of expression increases with advancing grade of malignancy. These findings support a role for Glut-1 in PC proliferation.

Increased discrimination between benign prostatic hyperplasia and prostate cancer through measurement of percentage free PSA.

OBJECTIVE: The value of Prostate-Specific Antigen (PSA) for the early detection of Prostate Cancer (CaP) is controversial due to an appreciable false positive rate causing unnecessary biopsies. As PSA exits in both free and bound forms the percentage of free PSA was found to be lower in CaP than in Benign Prostatic Hyperplasia (BPH). We investigated whether the percentage of free PSA offers better discrimination on the detection of CaP. MATERIAL AND METHODS: In a retrospective analysis the percentage of free PSA was determined in the sera of 50 consecutive patients with histologically proven BPH (n = 30) and clinically localised CaP without metastases (n = 20; pT1-3 No Mo). Serum levels of free PSA and total PSA were determined employing a chemiluminescent enzyme immunoassay. RESULTS: Patients with CaP demonstrated a lower percentage of free PSA (median: 8.5, range: 2.7-24.5) than patients with BPH (median: 22.35, range: 8.9-66.7). (p < 0.001). CONCLUSION: Determination of percentage of free PSA enhances the discrimination between BPH and CaP and may reduce the number of unnecessary biopsies in patients with elevated PSA.

[Prevention of catheter dislodgment: modified nephrostomy with pigtail and balloon]. / Prävention der Katheterdislokation. Modifizierte Nephrostomie mit Pigtail und Ballon.

Insertion of a percutaneous nephrostomy for decompression of the collecting system in cases of obstruction should be easy and safe to perform. Early or delayed catheter dislodgment is a more frequent event. Even with optimal positioning of the puncture site, this complication cannot be prevented reliably. Should dislodgment occur, a new puncture is necessary in most cases exposing the patient to risk and discomfort. The modified percutaneous nephrostomy tube presented here was designed for better protection against dislodgment while retaining technical ease of tube insertion.

[Clinical manifestations and indications for therapy of benign prostatic hyperplasia]. / Klinische Manifestation und Indikation zur Therapie der benignen Prostatahyperplasie.

The high incidence of benign prostatic hyperplasia (BPH), together with the wide variability in its clinical manifestations and in the natural course of disease, requires a careful evaluation of the patient. Technical progress and continuing development of established methods mean that a wide range of diagnostic procedures is available and an "objective" correlate of infravesical obstruction can be obtained. In addition to these objective criteria, the patient's subjective perception of the impact of the symptoms on his quality of life also affects the decision as to whether therapeutic intervention should be attempted and the degree of success that can be attained, as is becoming increasingly evident. The diagnostic methods available for the routine assessment of patients with BPH is reviewed, and their relative value as a basis for deciding on therapeutic intervention is analysed.

[Laparoscopic lymphadenectomy in prostate carcinoma. Experiences with 120 patients]. / Laparoskopische Lymphadenektomie beim Prostatakarzinom. Erfahrungen bei 120 Patienten.

Laparoscopic pelvic lymph node dissection was performed in 120 patients scheduled to undergo either radical perineal prostatectomy or radiation therapy. On average 13 lymph nodes were resected in each patient, and 21 patients were found to have metastatic disease in 1-9 lymph nodes. After an initial learning curve, mean operative time was reduced significantly, allowing laparoscopic lymph node dissection and radical prostatectomy to be performed as a one-stage procedure. The overall complication rate was 10%; open revisions were necessary in only 2 of 120 patients. Postoperative hospital stay was 2 days in patients undergoing laparoscopic lymph node dissection only. This minimally invasive procedure is particularly beneficial to patients with lymph node metastases not undergoing radical prostatectomy, as well as to patients planned to be treated by radiation therapy. The combination of laparoscopic lymph node dissection and radical perineal prostatectomy avoids an abdominal incision and thus shortens both the hospital stay and the period of convalescence.

Anemia under androgen deprivation: influence of flutamide, cyproteroneacetate and orchiectomy on the erythropoietin system.

OBJECTIVE: There is a high incidence of anemia in patients with advanced prostate cancer (PC) under androgen deprivation. Pathophysiology of this anemia remains unclear. Erythropoietin (EPO) is the main growth factor inducing erythropoesis in response to hypoxia. In this study, function of the EPO-system following androgen deprivation was tested in standardized animal model. METHODS: Animals were pretreated by either orchiectomy, injection of cyproteroneacetate or flutamide. After hypoxic stimulation, EPO mRNA expression and EPO serum levels were studied. RESULTS: In all animals, EPO mRNA expression and EPO serum levels were increased following hypoxic stimulation. Compared to the control group, this increase was even more pronounced after androgen deprivation. None of the different forms of androgen deprivation had a negative stimulating effect on EPO expression. CONCLUSION: Unexpectedly, androgen deprivation did not suppress EPO mRNA expression and EPO serum concentrations. Instead, stimulation of the EPO system was even more pronounced after androgen deprivation. A deficient EPO system does not appear to contribute to the clinically observed anemia in patients treated by androgen deprivation.

Basic research in prostate cancer: molecular biology.

While a general appreciation for the importance of chromosomes in the development of cancer has existed for decades, molecular genetic analyses have gained considerable attention in recent years through identification of proto-oncogenes and tumor suppressor genes. Several different chromosomal aberrations, alterations of proto-oncogenes and suppressor genes have been described in prostate cancer. Loss of genetic material has been found to occur most frequently on chromosomes 7, 8, 10 and 16. The existence of tumor suppressor genes relevant to prostate carcinogenesis is suspected in these chromosomal locations. Several investigators are currently trying to identify these genes. Altered expression of several different oncogenes has been reported in prostate cancer. Among these, the ras- and myc-families of oncogenes have been studied most intensively. Structural oncogene alterations have been detected infrequently, most of the changes appear to occur transcriptionally. Despite an abundance of clinical material, knowledge about genetic lesions in prostate cancer is still very limited and sometimes conflicting results have been reported. With recent methodologic improvements and a growing interest in correlating genetic alterations with clinical disease progression, definition of prostate carcinogenesis at the molecular level will advance rapidly in the near future.

Theories on the metastatic process and possible therapeutic options.

A sequence of steps are prerequisite for cancer cells before metastases are established. Metastasis has been shown to be an inefficient process limited by both random and selective events. By differentiating invasion from metastasis, sequential steps in the metastatic cascade have been defined and studied separately. This approach has yielded a variety of new potential therapeutic strategies. However, increasing knowledge of the mechanisms relating to metastasis has also revealed the complexity of each step. In spite of difficulties in translating results obtained in preclinical models into the clinical setting, continued development of such model systems and further research into the genetic control of metastatic dissemination will lead to improved strategies for prevention of metastasis formation and for treatment of metastatic tumor cells.

Laparoscopic pelvic lymphadenectomy.

We analyzed our first 41 patients with localized prostate cancer treated with laparoscopic pelvic lymph node dissection (LPLND) as staging procedure followed either by radical perineal prostatectomy or interstitial radiotherapy. 7/41 patients (17%) had pelvic lymph node metastases and received endocrine therapy. Overall complication rate was low (7.5%) and no major complication occurred. LPLND is minimally invasive and provides staging accuracy in prostate cancer.

Current and future strategies to block tumor angiogenesis, invasion, and metastasis.

Progression of malignancy involves a series of sequential steps that ultimately lead to cancer-cell dissemination. In addition to the loss of growth control, an imbalanced regulation of motility and proteolysis is a prerequisite for invasion and metastasis. These factors are also necessary for angiogenesis-an integral process occurring at both the primary and the metastatic sites. Investigators have elucidated in detail many of the molecular mechanisms involved in the sequential steps of the metastatic cascade and have thereby provided new targets for therapeutic intervention. For each step, different model systems have been developed and various strategies for antimetastatic therapy have been tested in vitro as well as in murine systems. Difficulties in translating results obtained in preclinical models into the clinical setting have become apparent and have not been unexpected in light of the sometimes highly artificial interaction in the experimental setting. Nevertheless, continued development of model systems and further research into the genetic control of malignancy should lead to the identification of common signal-transduction pathways. Interference at such sites promises to be particularly effective in inhibiting proliferation and metastasis.

Metabolic imaging of untreated prostate cancer by positron emission tomography with 18fluorine-labeled deoxyglucose.

PURPOSE: We evaluated positron emission tomograph (PET) with 18fluorine (18F)-labeled deoxyglucose for metabolic grading of untreated primary prostate cancer, and differentiation of benign and malignant prostatic disease. MATERIALS AND METHODS: A total of 48 patients with untreated prostate cancer of different stages and 16 with histologically confirmed benign prostatic hyperplasia (BPH) underwent static PET after intravenous injection of 150 to 300 MBq. 18F-deoxyglucose. 18F-deoxyglucose accumulation was quantitated by calculating differential uptake ratios and prostate-to-skeletal muscle ratios. RESULTS: Low 18F-deoxyglucose uptake was noted in the majority of primary tumors (81%). 18F-deoxyglucose accumulation did not correlate with increasing tumor grade or stage. There was a significant overlap in uptake values in BPH and malignant prostatic disease. A trend towards statistical significance was noted with lower prostate-to-skeletal muscle ratios in patients with BPH (p < 0.07). Increased 18F-deoxyglucose accumulation was detected in some patients with BPH and malignant prostatic disease, as well as in those with lymph node and bone metastases of prostate cancer. CONCLUSIONS: 18F-deoxyglucose PET does not allow for metabolic labeling in the majority of untreated primary prostate cancers. BPH and primary prostate cancer cannot be reliably differentiated on the basis of PET. Increased 18F-deoxyglucose accumulation occurs in some primary prostate tumors and in metastatic deposits of prostate cancer.

p53 gene alterations in human prostate carcinoma.

Alterations of the p53 gene are among the most frequent genetic lesions in a variety of human malignancies. Their role in prostate cancer is, however, unclear. We have analyzed 10 primary and two metastatic human prostate carcinomas as well as 3 prostate cancer cell lines for mutations of the p53-gene. Using single strand conformational polymorphism analysis (SSCP) and direct sequencing of the polymerase chain reaction (PCR) products, p53 mutations were detected in 1 of 10 primary prostate cancers. By contrast, 1 of 2 metastatic tumors and all 3 prostate cancer cell lines (derived from metastases) were found to contain a mutant p53 gene. Thus, our data suggest that alterations of the p53 gene at the mutational "hot spots" appear to occur infrequently in primary human prostate cancer, but may emerge in later stages of tumor progression. Furthermore, we confirm that loss of heterozygosity at a locus telomeric to p53, but not including p53, is associated with metastatic progression in 1 case.

Evaluation of patients with diseases of the prostate using prostate-specific antigen density.

OBJECTIVE: To compare the efficacy of two tests, prostatic-specific antigen (PSA) and the PSA/prostate volume ratio (PSAD), as diagnostic and staging markers to discriminate patients with benign prostatic hyperplasia (BPH) from patients with cancer of the prostate (CaP). PATIENTS AND METHODS: Prostate gland volumes were estimated in 60 patients with BPH and 88 patients with clinically organ-confined CaP by performing transrectal ultrasonography (TRUS) and using the prolate ellipse formula. Serum PSA concentration was determined using an enzyme immunoassay. In patients with BPH, the prostates were removed either by transurethral resection or retropubic prostatectomy. Patients with CaP underwent laparoscopic pelvic lymphadenectomy followed by radical perineal prostatectomy. PSAD was calculated by relating the serum PSA level to the TRUS-estimated prostate volume. RESULTS: The median PSA level was 4.4 ng/mL in patients with BPH, 9.3 ng/mL in patients with CaP-NO disease and 24 ng/mL in those with CaP-N+ disease. However, imposing a PSA limit of 4 ng/mL for the diagnosis of CaP gave a positive predictive value of only 64.8%, whereas a limit of 10 ng/mL gave a positive predictive value of 71.4%. In contrast, the median PSAD was 0.086 ng/mL/cm3 in patients with BPH, but was 0.295 ng/mL/cm3 in patients with NO-disease and 0.775 ng/mL/cm3 in those with N(+)-disease. With a limit of 0.15 ng/mL/cm3 the positive predictive value of PSAD was 81%. Furthermore, a limit of 0.6 ng/mL/cm3 revealed a positive predictive value of 81% for the diagnosis of metastatic lymph node involvement. CONCLUSIONS: There was a considerable overlap of PSA concentrations in patients with BPH and CaP, and PSA was not sufficiently accurate to distinguish between them. In contrast, PSAD enhanced the discrimination between BPH and CaP and may provide additional information about the status of the lymph nodes in patients with CaP.

Expression of the matrix Gla protein in urogenital malignancies.

Matrix Gla protein (MGP), is a vitamin-K-dependent protein which is synthesized in a variety of tissues such as lung, heart, kidney, cartilage and bone. The function of MGP in these tissues is unclear. We have previously reported elevated MGP mRNA levels in a breast-cancer cell line, 600PEI, as compared to normal breast epithelium. Here we describe high MGP expression in primary renal-cell carcinomas, prostate carcinomas and testicular germ-cell tumors, as determined by Northern analysis. MGP was over-expressed in 21 out of 28 patients with renal-cell carcinoma, and in 16 out of 29 patients with testicular germ-cell tumors, as compared to matched normal tissues. For the renal-cell carcinomas, a statistically significant inverse correlation was observed between the level of MGP expression and tumor size, lymph-node metastasis and tumor grade. MGP was also highly expressed in 13 primary prostatic carcinomas as compared to prostate cell lines derived from metastatic tumors, and to lymph-node metastasis. Our findings indicate that the loss of MGP expression may be associated wih tumor progression and metastasis.

Human papillomavirus types 16 and 18 are not involved in human prostate carcinogenesis: analysis of archival human prostate cancer specimens by differential polymerase chain reaction.

Human papilloma viruses (HPV) have been implicated in the pathogenesis of a variety of malignancies, especially in carcinomas of the female genital tract. Recently, based on observations using the polymerase chain reaction amplification assay, HPV types 16 and 18 specific DNA sequences have been detected in prostate cancer specimens obtained by transurethral resection. Since HPV types 16 and 18 have been shown to possess oncogenic potential, an association between HPV infection and prostatic carcinoma has been suggested. In order to exclude potentially HPV-colonized urethral mucosa from analysis and restrict our study to predominantly malignant tissue, cancerous areas from a series of 30 paraffin-embedded prostate adenocarcinomas were microdissected and analyzed for the presence of HPV 16 or HPV 18 specific sequences by a modification of PCR (D-PCR) and Southern blot analysis. Despite the high sensitivity of our analytical technique, we found no evidence of HPV-DNA of either type in any of the 30 primary prostate cancers. In contrast, both HPV 16 (2/8 specimens) and HPV 18 (2/8 specimens) DNA was detected in randomly chosen cervical carcinomas using the D-PCR methodology. Our data would indicate that the oncogenic HPV-types 16 and 18 are unlikely effectors of prostate carcinogenesis.

Alterations of the P53 gene are associated with the progression of a human prostate carcinoma.

P53 is a tumor suppressor gene that has been implicated in the molecular genetics of many human malignancies. Nucleotide alterations, most commonly single point mutations, have been shown not only to abrogate the p53 suppressor function but also to contribute to the transformed phenotype. We report the detection of a p53 gene mutation in clinical specimens of a patient with relapsing prostate adenocarcinoma 14 years after definitive external beam radiation. The techniques of single strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction generated products were used for this study. Analysis of tissue from different locations of the primary tumor revealed intratumoral molecular heterogeneity; the mutation was absent in 1 area but present in another. Tumor from a regional lymph node metastasis harbored the identical p53 mutation. Furthermore, an additional genetic alteration, an allelic loss on chromosome 17p but not including the p53 gene, was observed only in the metastatic tissue. These observations in clinical specimens of primary and metastatic sites provide evidence for the association of the p53 gene in the progression of human prostate carcinoma.