Alternative splicing of TGF-betas and their high-affinity receptors T beta RI, T beta RII and T beta RIII (betaglycan) reveal new variants in human prostatic cells.

BACKGROUND: The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2 and TGF-beta 3, and their receptors [T beta RI, T beta RII, T beta R III (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants T beta RIB, T beta RIIB and TGF-beta 2B in human prostatic cells. RESULTS: Interestingly, a novel human receptor transcript T beta RIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant T beta RIB with four additional amino acids was identified also in human. Expression of the variant T beta RIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of T beta RIIC and TGF-beta 2B mainly in the epithelial cells with a preferential localization of TGF-beta 2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-beta ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-beta2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of T beta RII. CONCLUSION: In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants T beta RIB and the novel isoform T beta RIIC in man clearly contributes to the growing complexity of the TGF-beta family.

Genetic pathways and new progression markers for prostate cancer defined by microsatellite allelotyping.

A prospective study was carried out on a large cohort of males undergoing radical retropubic prostatectomy in order to identify genetic marker regions significantly associated with tumor formation. By comprehensive allotyping of chromosomes known to be associated with prostate carcinogenesis, an algorithm could be formulated for the genetic pathway and a method of discrimination between aggressive and less aggressive forms could be identified.

Molecular serological diagnosis in transitional cell bladder cancer.

There are no reliable serological tumor markers for transitional cell carcinoma (TCC) of the urinary bladder. Fluorescent microsatellite analysis (MSA) was used to detect serum-DNA alterations in patients with bladder cancer. Prospectively, fresh tumor-, peripheral blood-, and serum-specimens were collected from 58 consecutive patients treated for TCC of the bladder to obtain the corresponding DNA. DNA was extracted by the phenol-chloroform method from tumors and blood lymphocytes. Serum DNA was isolated by a commercial kit. Fluorescent MSA was performed with a total of 17 polymorphic markers from chromosomal regions 5q, 8p, 9p, 9q, 13q, 14q, 17p, 17q, and 20q in the 58 cancer specimens as well as in specimens from 20 healthy controls. Detection of allelic imbalance and loss of heterozygosity in the tumor and serum specimens was carried out on an automated laser sequencer. Serum-DNA alterations were identified in 79.3% (46/58). Four healthy controls displayed serum-DNA artefacts rendering a specificity of 80%. The highest frequency of serum-DNA alterations (38%) was detected for chromosomal region 8p. Chromosomes 5q, 9p, and 20q showed serum-DNA alterations in 20% to 23%. Identification of tumor-specific serum-DNA alterations was stage independent (P >0.05), but was more frequent in high-grade tumors (P = 0.08). To optimize specificity, simultaneous analysis of tumor DNA is advised to rule out artefacts resembling allelic imbalance in MSA of serum DNA. The method then has potential for application as a "serological marker" in the follow-up of patients after radical surgical therapy for invasive TCC of the bladder.

Molecular serological detection of DNA alterations in transitional cell carcinoma is highly sensitive and stage independent.

PURPOSE: To evaluate the efficacy of fluorescent microsatellite analysis (MSA) for the serological diagnosis of transitional cell carcinoma (TCC) of the urinary tract analyzing free tumor DNA in the serum of cancer patients. EXPERIMENTAL DESIGN: We applied fluorescent MSA to detect serum-DNA alterations in patients suffering from bladder and upper urinary tract TCC and prospectively collected fresh tumor, peripheral blood, and serum specimens from 61 consecutive patients to obtain the corresponding DNA. Fluorescent MSA was performed with a total of 17 polymorphic markers from the chromosomal regions 5q, 8p, 9p, 9q, 13q, 14q, 17p, 17q, and 20q in the 61 cancer patients, as well as in 20 healthy controls. RESULTS: Molecular serological analysis led to tumor-specific diagnosis of TCC in 80.3% (49 of 61) of cases. Four healthy controls displayed serum-DNA artifacts rendering a specificity of 80%. The highest frequency of serum-DNA alterations was detected for chromosomal region 8p with 36%. Chromosomes 5q, 9p, and 20q showed serum-DNA alterations in 18 to 21%. The identification of serum-DNA alterations was not statistically associated with underlying local tumor stage (P = 0.29) but was more frequent in high-grade tumors (P = 0.08). CONCLUSIONS: MSA offers a highly sensitive method for serological diagnosis of TCC. To optimize specificity, simultaneous analysis of tumor DNA is advised to rule out artifacts resembling allelic imbalance in MSA of serum DNA.

Genetic pathways and new progression markers for prostate cancer suggested by microsatellite allelotyping.

PURPOSE: At diagnosis, the biological behavior of prostate cancer is uncertain, making the choice of an adequate therapy option difficult. Performing microsatellite allelotyping on a large series of consecutive prostate cancers procured during radical prostatectomy at our institution, we sought to identify molecular markers associated with disease progression. EXPERIMENTAL DESIGN: A total of 156 consecutive fresh tumor samples was prospectively collected and macroscopically dissected from the whole prostatectomy specimen immediately after operation. Histologically 100 samples contained >75% tumor cells and were therefore enrolled in the microsatellite allelotyping, using a total of 24 polymorphic markers for the chromosomal regions 5p, 5q, 7q, 8p, 9p, 9q, 13q, 17p, 17q, and 18q. Fresh paired normal and tumor DNA was investigated in fluorescent microsatellite analysis with automated laser product detection. RESULTS: The incidence of tumor-DNA alterations [loss of heterozygosity or allelic imbalance (AI)] was highest for chromosomal regions 13q and 8p with 72 and 71%, respectively, followed by chromosomes 7q, 18q, 5q, and 17p with 57, 53, 41, and 39%, respectively. Alterations at chromosomes 8p, 9p, 13q, and 17p were significantly (P < 0.05) associated with advanced tumor stage, whereas AI at 8p and 17p was also associated with high Gleason score (P < 0.05). AI at 5q and 9p was associated with regional lymph node metastasis (P < 0.05). The combination of AI at 8p and 13q was strongly associated with advanced tumor stage (P < 0.0001). CONCLUSIONS: With the obtained results, we are able to postulate three distinct pathways in prostate carcinogenesis, and we identified microsatellite markers of prognostic value.

High frequency of serum DNA alterations in renal cell carcinoma detected by fluorescent microsatellite analysis.

To date there are no reliable serological markers for renal cell carcinoma (RCC). We applied fluorescent microsatellite analysis (MSA) to detect serum DNA alterations in patients with RCC. Fresh tumour, peripheral blood and serum specimens from 60 consecutive patients treated for malignant renal tumours (n= 53 RCC and n= 7 non-RCC) were prospectively collected. After DNA extraction, we performed MSA with a total of 9 markers from the chromosomal regions 3p, 5q, 7p, 7q, 9p, 13q, 17p and 17q to identify tumour specific serum DNA alterations in Group I (n= 53 RCC); 11 additional markers were used in the first 23 RCCs (Group II) in order to increase sensitivity; and 20 healthy controls were investigated with 10 markers. Besides the histomorphological diagnosis the RCCs were genetically stratified according to the "Heidelberg Classification" of renal tumours. Detection of allelic imbalance and loss of heterozygosity (LOH) was carried out on an automated laser sequencer. In Group I we identified serum DNA alterations in 74% (39/53) of cases. When applying 20 markers, the sensitivity was elevated to 87% (20/23) in Group II. Investigating 20 healthy controls with 10 markers, the method rendered 85% specificity. The highest incidence of alterations was detected for chromosomal regions 3p and 5q. The presence of serum DNA alteration was not associated with tumour nuclear grade but exhibited a trend towards advanced stages (p = 0,044). In RCC, the microsatellite analysis has a high sensitivity in the detection of serum DNA alterations when a sufficient number of markers from various chromosomal regions are used. Advanced tumours tend to express serum DNA alterations more frequently.