Automatic lung segmentation in COVID-19 patients: Impact on quantitative computed tomography analysis.

PURPOSE: To assess the impact of lung segmentation accuracy in an automatic pipeline for quantitative analysis of CT images. METHODS: Four different platforms for automatic lung segmentation based on convolutional neural network (CNN), region-growing technique and atlas-based algorithm were considered. The platforms were tested using CT images of 55 COVID-19 patients with severe lung impairment. Four radiologists assessed the segmentations using a 5-point qualitative score (QS). For each CT series, a manually revised reference segmentation (RS) was obtained. Histogram-based quantitative metrics (QM) were calculated from CT histogram using lung segmentationsfrom all platforms and RS. Dice index (DI) and differences of QMs (ΔQMs) were calculated between RS and other segmentations. RESULTS: Highest QS and lower ΔQMs values were associated to the CNN algorithm. However, only 45% CNN segmentations were judged to need no or only minimal corrections, and in only 17 cases (31%), automatic segmentations provided RS without manual corrections. Median values of the DI for the four algorithms ranged from 0.993 to 0.904. Significant differences for all QMs calculated between automatic segmentations and RS were found both when data were pooled together and stratified according to QS, indicating a relationship between qualitative and quantitative measurements. The most unstable QM was the histogram 90th percentile, with median ΔQMs values ranging from 10HU and 158HU between different algorithms. CONCLUSIONS: None of tested algorithms provided fully reliable segmentation. Segmentation accuracy impacts differently on different quantitative metrics, and each of them should be individually evaluated according to the purpose of subsequent analyses.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

BACKGROUND: DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. METHODS: 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. RESULTS/CONCLUSIONS: Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.

SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses.

BACKGROUND: The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. METHODS: SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. RESULTS: 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. CONCLUSIONS: The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of DNA analysis of blood samples.

SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1ß, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples.

In vivo effects of rosiglitazone in a human neuroblastoma xenograft.

BACKGROUND: Neuroblastoma (NB) is the most common extra-cranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. There is in vitro evidence that the peroxisome proliferator-activated receptor gamma (PPARgamma) might be a target for pharmacological intervention in NB. We have previously demonstrated that the PPARgamma agonist rosiglitazone (RGZ) exerts strong anti-tumoural effects in the human NB cell line, SK-N-AS. The aim of this study was to evaluate whether RGZ maintains its anti-tumoural effects against SK-N-AS NB cells in vivo. METHODS AND RESULTS: For this purpose, tumour cells were subcutaneously implanted in nude mice, and RGZ (150 mg kg(-1)) was administered by gavage daily for 4 weeks. At the end of treatment, a significant tumour weight inhibition (70%) was observed in RGZ-treated mice compared with control mice. The inhibition of tumour growth was supported by a strong anti-angiogenic activity, as assessed by CD-31 immunostaining in tumour samples. The number of apoptotic cells, as determined by cleaved caspase-3 immunostaining, seemed lower in RGZ-treated animals at the end of the treatment period than in control mice, likely because of the large tumour size observed in the latter group. CONCLUSIONS: To our knowledge, this is the first demonstration that RGZ effectively inhibits tumour growth in a human NB xenograft and our results suggest that PPARgamma agonists may have a role in anti-tumoural strategies against NB.

Androgen-responsive and -unresponsive prostate cancer cell lines respond differently to stimuli inducing neuroendocrine differentiation.

The treatment of advanced prostate cancer (CaP) with androgen deprivation therapy inevitably renders the tumours castration resistant and incurable. Under these conditions, neuroendocrine differentiation (NED) of CaP cells occurs and neuropeptides released by neuroendocrine cells facilitate tumour progression. Pharmacological strategies aiming to prevent or delay NED during androgen ablation could, therefore, increase the effectiveness of the therapy. Mechanisms and pathways inducing NED in CaP are poorly understood and data are often discordant. In the present study, we used several CaP cell lines (androgen-responsive: LNCaP, PC3-AR, 22RV1 and -irresponsive: DU145 and PC3) to evaluate NED after androgen deprivation or treatment with epidermal growth factor (EGF). NED was determined by neuron-specific enolase and chromogranin A expression and by the occurrence of morphological changes in the cells. Androgen-deprivation conditions induced NED in LNCaP and PC3-AR, but not in 22Rv1, PC3 and DU145 cells. LNCaP and PC3-AR cells also became resistant to thapsigargin-induced apoptosis. In all the AR-positive cell lines, androgen deprivation caused a decrease in androgen receptor expression indicating that it is downregulated irrespective of NED induction. Treatment with EGF induced NED in DU145 cells and the EGF receptor inhibitor gefinitib prevented the process. On the contrary, no effect of EGF was demonstrated in LNCaP or 22Rv1 cells. CaP cell lines did not respond univocally to treatments inducing NED, suggesting that studies on this topic should be performed in a wide spectrum of cell models which can be more indicative of the tumour variability in vivo.

The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis.

Chemokines exert their multifunctional role in several physiologic and pathologic processes through interaction with their specific receptors. Much evidence have revealed that metastatic spread tumor cells may use chemokine-mediated mechanisms. In particular, an involvement of stromal cell-derived factor-1 (SDF-1) in growth of primary tumors and in metastatic process has been demonstrated. Indeed, it has been suggested that CXCR4 expression by tumor cells, plays a critical role in cell metastasis by a chemotactic gradient to organs expressing the ligand SDF-1. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. In physiologic condition, SDF-1 also plays an essential role modulating stem cell proliferation, survival, and homing through its canonical receptor CXCR4. Recently, several studies have demonstrated the existence of a small subset of cancer cells which share many characteristics with stem cells and named cancer stem cells (CSC). They constitute a reservoir of self-sustaining cells with the ability to maintain the tumor growth. In particular, most of them express CXCR4 receptor and respond to a chemotactic gradient of its specific ligand SDF-1, suggesting that CSC probably represent a subpopulation capable of initiating metastasis. This review focuses on the role of SDF-1/CXCR4 axis in cancer and in the metastatic progression by tumoral cells, as well as the role of CSC in tumor pathogenesis and in metastatic process. A better understanding of migratory mechanism involving cancer cells and CSC provides a powerful tool for developing novel therapies reducing both local and distant recurrences.

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells.

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.

Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.

Analysis of somatostatin receptors 2 and 5 polymorphisms in patients with acromegaly.

OBJECTIVE: The aim of the study was to investigate the possible correlation of single nucleotide polymorphisms in somatostatin receptor (SSTR)2 and SSTR5 genes with the responsiveness to somatostatin analogs in a cohort of acromegalic patients. STUDY DESIGN: Three single nucleotide polymorphisms (a-83 g, c-57 g, and t80c) of SSTR2 and three (t-461c, c325t, and c1004t) of SSTR5 were analyzed in 66 acromegalic patients with different responsiveness to somatostatin analogs and 66 healthy controls. RESULTS: Allele frequencies in patients and controls were similar. No association between SSTR2 genotypes and GH and IGF-I levels was found. When considering SSTR5 variants, patients homozygous or heterozygous for the substitution c1004 (P+) showed basal IGF-I levels significantly lower than patients homozygous for 1004t (P-). Moreover, serum GH levels were lower in patients with P+/T- haplotype (having c1004 allele and no t-461 allele) than in those with P-/T+. No correlation between SSTR2 and SSTR5 genotypes, responsiveness to somatostatin therapy, and mRNA expression in the removed adenomas (n = 10) was found. CONCLUSIONS: These data suggest a role for SSTR5 t-461c and c1004t alleles in influencing GH and IGF-I levels in patients with acromegaly, whereas SSTR2 and SSTR5 variants seem to have a minor role in determining the responsiveness to somatostatin analogs.

Measurement of somatostatin receptor subtype 2 mRNA in breast cancer and corresponding normal tissue.

Somatostatin analogs are effective in inhibiting growth of human breast cancer cell lines. These antiproliferative effects are mediated by specific receptors located on cell membranes. The somatostatin receptor subtype 2 (sst2) is the principal mediator of somatostatin effects in normal and cancer cells, and its presence has already been demonstrated in breast cancer. The purpose of our study was to evaluate the clinical relevance of the expression of sst2 by quantifying its mRNA in a large group of infiltrating breast cancers and their corresponding normal tissues. The expression of sst2 mRNA was measured with quantitative real time RT-PCR in 169 breast cancers and in their corresponding unaffected tissues. We evaluated the association of sst2 expression with the commonest clinical-pathologic features of breast cancer. The correlation with a marker of cell proliferation (Ki-67) and with receptor concentration was also evaluated. In cancer tissues, we found that the absolute concentrations of sst2 mRNA were significantly higher in estrogen receptor (ER)-positive samples (P=0.002) as well as in lymph-node-negative cancers (P=0.04) (Student's t-test or one-way ANOVA). In addition, sst2 mRNA was significantly higher in breast cancers than in corresponding unaffected tissues (P=0.0002). However, when the clinical-pathologic parameters were considered, this gradient maintained its statistical significance only in tumors expressing positive prognostic markers, such as the presence of ER (P=0.0005) and progesterone receptors (PgR) (P=0005), and the lack of lymph-node involvement (P=0.0003). The same difference was also significant in postmenopausal women (P=0.001) and in T1 patients (P=0.001). In addition, sst2 mRNA expression was significantly higher (P=0.008) in low-proliferating breast cancers. Finally, we found that the quantitative expression of sst2 mRNA was directly related to the PgR concentration in breast cancer tissues (P<0.001). Our data seem to indicate that an upregulation of sst2 gene expression is a common feature of breast cancers which, on the basis of conventional predictive parameters, are expected to have a better prognosis. Featuring a possible role of somatostatin analogs in combined endocrine therapies for breast cancer, our results seem to confirm that the sst2 status of the tumor should be previously investigated.

Inhibition of prostate cell growth by BXL-628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia.

OBJECTIVE: Calcitriol analogues might represent an interesting new therapy for benign prostate hyperplasia (BPH). We here report the preclinical characterization of BXL-628, an analogue selected for an ongoing double-blind, randomized, placebo-controlled phase II trial in BPH. DESIGN: Experiments with BXL-628 were carried out in human BPH cells and in the ventral prostate of intact and castrated rats. METHODS: BPH cell and rat prostate growth were evaluated along with morphological and biochemical hallmarks of apoptosis. RESULTS: BXL-628 inhibited human BPH cell proliferation and induced apoptosis even in the presence of androgens or growth factors. It also decreased prostate growth to an extent similar to finasteride, inducing DNA fragmentation and apoptosis, both in intact and in testosterone-supplemented castrated rats. Accordingly, BXL-628, like finasteride, increased the expression of clusterin, a prostatic atrophy marker. However, BXL-628 did not inhibit 5 alpha-reductase 1 and 2, did not bind to the androgen receptor (AR) in BPH homogenates and did not affect AR-coupled luciferase activity. In addition, BXL-628 did not affect rat pituitary and testis activity or calcemia. CONCLUSIONS: BXL-628 inhibited in vitro and in vivo prostate cell proliferation, and therefore might represent a novel, interesting option for the treatment of BPH.

Loss of heterozygosity at the SS receptor type 5 locus in human GH- and TSH-secreting pituitary adenomas.

SS receptor types 2 and 5 (sst2 and sst5) are involved in the control of secretion and proliferation of normal and tumoral somatotrophs and thyrotrophs. The mechanisms leading to reduced responsiveness to SS analogues in patients with pituitary tumors are poorly understood. The aim of the study was to verify the possible loss of heterozygosity (LOH) at the sst5 gene locus in somatotroph and thyrotroph adenomas by screening leukocyte and tumor DNA for two single nucleotide polymorphisms, i.e. C1004T leading to P335L change and T-461C in the 5'-upstream region. Among the 13 informative samples, 1 GH- and 1 TSH-secreting adenoma showed LOH at sst5 gene locus with the retention of Leu335 variant. By analyzing other polymorphic markers spanning from telomere to 16p13.3-13.2 boundaries, DNA deletion of at least 1 megabase was found in both tumors. LOH in thyrotroph adenoma was associated with unusual tumor aggressiveness that required a second surgery and resistance to SS analogs, while no obvious phenotype was identified in the case of the somatotroph adenoma. In conclusions, LOH at the sst5 gene locus is a rare phenomenon, occurring in about 10% of pituitary tumors, that seems to be associated with an aggressive phenotype, at least in thyrotroph adenomas. Further studies are required to confirm this association and to identify the genes, in addition to sst5, lost in these tumors.

[Somatostatin receptors in non-endocrine tumours]. / Recettori per la somatostatina in tumori non endocrini.

The study of the antiproliferative action of somatostatin (ss) is important not only to understand the regulation of neuroendocrine tumours that express receptors (sst), but also non-endocrine tumours which express these receptors. We previously demonstrated the presence of sst2 in a wide panel of cell lines from human neuroblastoma. Although hypotheses have been put forward that treatment with ss or its analogs may be beneficial in oncological patients, this does not appear to be the case in neuroblastoma; patients with high sst2 levels (who are therefore sensitive to ss treatment) have per se a relatively positive outcome. Therefore, adjuvant treatment with ss is not necessary. Viceversa, patients with a poor prognosis are essentially characterized by a low expression of sst2 (and therefore are insensitive to a therapy with ss). In these patients adjuvant treatment with ss might be indicated, but would have little chance of success. Although the majority of neuroendocrine tumours expresses sst2, pancreas and prostate cancer express sst1 but not sst2, and are therefore insensitive to octreotide treatment which binds preferentially to sst2. Tumours like colorectal carcinoma and breast cancer also express sst2 in their more favourable forms. However, the concentration of sst2 in colorectal cancer is similar, if not lower than that in the surrounding normal tissue. Therefore, the probability of successful adjuvant therapy with ss is relatively low. In breast cancer, it is possible that sensitivity to estrogens may have a positive influence on the expression of sst2. This might justify clinical trials with ss in breast cancer.

Variable expression of the transcription factors cAMP response element-binding protein and inducible cAMP early repressor in the normal adrenal cortex and in adrenocortical adenomas and carcinomas.

The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms alpha, beta, gamma, and tau2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.

Telomerase in endocrine and endocrine-dependent tumors.

Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.

Telomerase in urological malignancy.

PURPOSE: Telomerase is a ribonucleoprotein enzyme that compensates for the progressive erosion of chromosomal ends, called telomeres. In most somatic cells telomerase expression is repressed and telomeres progressively shorten after each cell division, causing cell senescence. Conversely telomerase is active in most human cancers, maintaining the integrity of chromosome ends and representing an important step in cell immortalization and carcinogenesis. The large and increasing interest in telomerase was motivated by the demonstration that more than 90% of human cancers are telomerase positive, whereas most normal tissues or benign tumors contained low or undetectable telomerase activity. We addressed the most recent data on telomerase detection in urological malignancy. Approaches to telomerase inhibition as a future anti-cancer therapy are also discussed. MATERIALS AND METHODS: We comprehensively reviewed the most recent and significant publications in this field using current issues of specific journals and a MEDLINE search. RESULTS: Telomerase is often expressed in bladder (90%), prostate (80%) and renal (69%) carcinoma. A variable but significant percent of normal tissues from tumor adjacent zones or noncancer samples are positive for telomerase. The clinical role of telomerase is still questionable in renal cancer, while important insights into the diagnostic role of telomerase in bladder and prostate carcinoma are increasing. Telomerase detection in exfoliated cells collected with urine or bladder washings seems a promising tool for the diagnosis and management of bladder cancer. CONCLUSIONS: Larger perspective studies of larger groups of patients are required to discover an appropriate role for telomerase when assessing these tumors. The improvement of quantitative methods to evaluate the expression of telomerase is a cornerstone in the complete clarification of the clinical relevance of telomerase.

Real-Time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for the measurement of prostate-specific antigen mRNA in the peripheral blood of patients with prostate carcinoma using the taqman detection system.

Circulating prostate cells can be detected in peripheral blood of patients with clinically localized or advanced prostate carcinoma. Traditionally, nested reverse transcriptase-polymerase chain reaction (RT-PCR) is used for this as a sensitive, but qualitative only, detection system. We developed a quantitative real-time RT-PCR method for measuring prostate-specific antigen (PSA) mRNA in peripheral blood of prostate cancer patients. A quantitative assay was developed using an external standard reference curve generated with RNA from the human prostate cell line LNCaP. Basal blood samples were collected from 44 patients without evidence of distant metastases and from 30 healthy controls. In 29 patients surgically treated with radical prostatectomy, the measurement of PSA mRNA was performed in blood samples collected before, at the end and 6 days after surgery. In 14 patients treated with radiotherapy, the measurements were repeated at 3-month intervals to evaluate time-related changes during therapy. The measurements were also performed for one year at 3-month intervals in one patient treated with anti-androgen therapy. We found detectable PSA mRNA in 14/44 (32%) basal blood samples. A wide range of values were observed in these patients, ranging from 0.5 to 1724 pg of total LNCaP RNA/ml blood. In patients undergoing radical prostatectomy, circulating PSA mRNA was detectable in eight patients in basal samples, and in seven of them also in blood specimens collected at the end of surgery, showing an increase in only two patients. In blood samples collected 6 days later, PSA mRNA was dramatically reduced in all patients, but still present in seven of them. In four patients, whose basal samples were negative, PSA mRNA was detectable in samples collected at the end of surgery and three of them were negative after 6 days. In patients who did not receive surgical treatment, a rapid decrease in PSA mRNA was demonstrated in five patients treated with radiotherapy and in one patient undergoing androgen deprivation. No detectable PSA mRNA was found in healthy controls. The levels of PSA mRNA in peripheral blood from patients with prostate carcinoma can be easily measured by this sensitive, quantitative and reliable procedure. This assay is a promising tool for the detection and follow-up of these patients.

Evidence for elevated telomerase activity in small cell carcinoma of the bladder.

Telomerase activity was found to be elevated using a quantitative assay on snap-frozen protein extracts of exfoliated cells in urine and bladder washings and tumor tissue obtained from a male patient with small cell carcinoma of the bladder. To the best of our knowledge, this is the first demonstration of elevated values of telomerase activity in genitourinary small cell carcinoma and is in keeping with the findings in primary lung locations.