Identification of circulating prostate cancer cells: a challenge to the clinical implementation of molecular biology (review).

Conventional diagnosis of prostate cancer does not appear to be sensitive enough to differentiate pre-operatively between organ-confined and extracapsular disease. New technologies, arising from the field of molecular biology, have been introduced to improve diagnosis and their implementation into the clinical practice is nowadays extensively explored. In 1992, focusing on prostate cancer, the application of the highly sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) technology which amplifies predefined mRNA species was introduced. Assuming haematogenous prostate tissue-specific mRNA species to be representative for the presence of circulating prostate cancer cells, an impressive series of clinical studies, for the greater part addressing mRNA encoding for prostate-specific antigen (PSA), were performed to improve pre-operative staging (molecular staging) and prognosis of prostate cancer, and to study iatrogenic cell dissemination. In this review we summarize the efforts, concentrating on the RT-PCR methodology, to identify extracapsular prostate cancer cells in easy accessible body fluids. The substantial amount of available biological and clinical data allow an in depth illustration of the advantages, disadvantages and clinical future of this technology. The intrinsic limitations of the technology, technical as well as biological, will be addressed since these may well explain the controversies associated with its general acceptance in the clinical practice. Together with considerable improvements of the methodology to be expected in the near future, new avenues for the detection of disseminated prostate cancer cells will be subject of discussion.

Bone resorption and renal calcium reabsorption in renal cell carcinoma-bearing mice: the effects of bisphosphonate.

OBJECTIVE: To examine the contribution of the skeleton and the kidney to the development of humoral hypercalcaemia of malignancy (HHM) in a mouse model of HHM treated with a potent bisphosphonate. MATERIALS AND METHODS: Mice bearing the human RCC cell line RC-9 were treated with bisphosphonate (subcutaneous, 0.25 mg/kg body weight olpadronate) or saline solution. Treatment was initiated at a tumour volume (TV) of approximately 100 mm(3) and 500 mm(3), and the mice were monitored for approximately 4 weeks. Serum calcium and phosphate concentrations and trabecular bone volume (TBV) were assessed during and/or after treatment. RESULTS: Athymic mice implanted with the RCC RC-9, developed severe hypercalcaemia and bone resorption. During tumour growth the mean (sd) serum calcium concentration increased to 4.1 (0.3) mmol/L, and phosphate decreased to 1.6 (0.3) mmol/L, vs 2.3 (0.1) and 2.9 (0.4) mmol/L in controls, respectively. TBV decreased from 8.7 (1.8)% in mice with no tumour, to 5.3 (2.7)% in RC-9-bearing mice. Olpadronate initiated at a Tv of 100 mm(3) prevented the loss of bone induced by RCC RC-9 cells, with a TBV of 12.8 (2.1)%, but the development of hypercalcaemia was unaffected. Olpadronate treatment at a TV of 500 mm(3) did not influence the development of hypercalcaemia and did not protect against bone resorption. Kinetic monitoring showed an identical rate of tumour growth in the presence or absence of bisphosphonate, while under both conditions there was a tumour load-dependent increase in calcium concentration. CONCLUSIONS: Bisphosphonate can prevent parathyroid hormone-related peptide (PTHrP)-mediated bone resorption when administered during the early phase of renal tumour growth, but has no effect on the tumour-induced development of hypercalcaemia, indicating a primary role for renal tubular reabsorption of calcium in the kidney by PTHrP in HHM.

BCG dose reduction by decreasing the instillation frequency: effects on local Th1/Th2 cytokine responses in a mouse model.

OBJECTIVES: Based on the requirement of a Th1 immune response for clinical efficacy, and incited by the arbitrary induction scheme, frequent side effects and the empirical approach in improving BCG immunotherapy for superficial bladder cancer, an alternative intravesical BCG treatment schedule for dose reduction was investigated without compromising Th1 cytokine induction in the bladder in a mouse model. METHODS: Mice were submitted to 6 weekly BCG instillations and treatment schedules omitting intermediate instillations during this standard scheme. Th1 (IFN-gamma, IL-2, IL-12p40), and Th2 (IL-10, IL-4) cytokine responses in individual mouse bladders were measured by a semiquantitative RT-PCR based method. RESULTS: A schedule of only two BCG instillations, administered in week 1 and week 6, resulted in induction of at least the same levels of IFN-gamma, IL-2 and IL-12p40 Th1 cytokine mRNA compared to 6 weekly instillations, whereas significantly lower levels of Th2 cytokines IL-10 and IL-4 mRNA were observed. CONCLUSIONS: During the 6-week period the intermediate weekly BCG instillations 2, 3, 4, and 5 do not contribute to Th1 cytokine upregulation in the bladder, provided that the BCG dose is sufficient. Whether such a reduced BCG frequency schedule has immune stimulating capacity and therapeutic efficacy associated with less side effects in patients remains to be investigated.

Cytokine gene expression in a mouse model: the first instillations with viable bacillus Calmette-Guerin determine the succeeding Th1 response.

PURPOSE: Bacillus Calmette-Guerin (BCG) therapy for superficial bladder cancer is immune dependent and activation of a Th1 immune response is probably required for clinical efficacy. Given the empirical approach to improving BCG therapy we investigated in a mouse model the consequences of modifications in BCG therapy with regard to Th1 and Th2 cytokine responses in the bladder. These studies may provide a rationale for possible modifications of the established clinical treatment protocol. MATERIALS AND METHODS: The dynamics of Th1 (interferon-gamma, interleukin [IL]-2, IL-12p40 and tumor necrosis factor-alpha) and Th2 (IL-10 and IL-4) cytokine responses during and after 6 once weekly intravesical BCG instillations in mice was determined by a semiquantitative reverse transcriptase-polymerase chain reaction based method. RESULTS: During 6 weekly BCG instillations a dose and time dependent induction of the various Th1 as well as Th2 cytokines was observed. The response pattern was comparable to urinary cytokine induction patterns in patients. Electrocauterization prior to BCG instillations led to lower and more variable levels of cytokine polymerase chain reaction products compared with noncauterization. Lowering the dose of BCG seemed to affect the Th1 cytokine response most, whereas the Th2 response was less influenced by dilution of the BCG preparation. Six instillations with nonviable BCG induced Th2 but failed to induce Th1 cytokines, which may explain the necessity of BCG viability for antitumor activity. However, when mice were first treated 3 times with viable BCG and subsequently received 3 instillations with killed BCG, the Th1 and Th2 cytokine pattern was comparable to the standard 6-week regimen with viable BCG. CONCLUSIONS: The model seems an appropriate one in which to investigate changes in Th1 and Th2 cytokine gene expression levels in bladders resulting from modifications in intravesical BCG treatment. It was possible to induce a local Th1 cytokine response with nonviable BCG provided that local sensitization to BCG antigens had occurred during preceding instillations with a viable BCG preparation. Whether such an approach could decrease BCG therapy toxicity, while maintaining antitumor efficacy, remains to be further investigated in patients.

Numeric definition of the clinical performance of the nested reverse transcription-PCR for detection of hematogenous epithelial cells and correction for specific mRNA of non-target cell origin as evaluated for prostate cancer cells.

BACKGROUND: Inappropriate quality management of reverse transcription-PCR (RT-PCR) assays for the detection of blood-borne prostate cancer (PCa) cells hampers clinical conclusions. Improvement of the RT-PCR methodology for prostate-specific antigen (PSA) mRNA should focus on an appropriate numeric definition of the performance of the assay and correction for PSA mRNA that is not associated with PCa cells. METHODS AND RESULTS: Repeated (RT-)PCR tests for PSA mRNA in single blood specimens from PCa patients and PCa-free controls, performed by four international institutions, showed a large percentage (approximately equal to 50%) of divergent test results. The best estimates of the mean, lambda (SD), of the expected Poisson frequency distributions of the number of positive tests among five replicate assays of samples from PCa-free individuals were 1.0 (0.2) for 2 x 35 PCR cycles and 0.2 (0.1) for 2 x 25 PCR cycles. Assessment of the numeric value of the mean can be considered as a new indicator of the performance of a RT-PCR assay for PSA mRNA under clinical conditions. Moreover, it determines the required number of positive test repetitions to differentiate between true and false positives for circulating prostate cells. At a predefined diagnostic specificity of > or = 98%, repeated PCRs with lambda of either 1.0 or 0.2 require, respectively, more than three or more than one positive tests to support the conclusion that PSA mRNA-containing cells are present. CONCLUSIONS: Repeated nested PCR tests for PSA and appropriate handling of the data allow numeric quantification of the performance of the assay and differentiation between analytical false and true positives at a predefined accuracy. This new approach may contribute to introduction of PSA RT-PCR assays in clinical practice.