Serum levels of an isoform of apolipoprotein A-II as a potential marker for prostate cancer.

PURPOSE: We recently showed that protein expression profiling of serum using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) has potential as a diagnostic approach for detection of prostate cancer. As a parallel effort, we have been pursuing the identification of the protein(s) comprising the individual discriminatory "peaks" and evaluating their utility as potential biomarkers for prostate disease. EXPERIMENTAL DESIGN: We employed liquid chromatography, gel electrophoresis and tandem mass spectroscopy to isolate and identify a protein that correlates with observed SELDI-TOF MS mass/charge (m/z) values. Immunodepletion, immunoassay, and Western analysis were used to verify that the identified protein generated the observed SELDI peak. Subsequent immunohistochemistry was used to examine the expression of the proteins in prostate tumors. RESULTS: An 8,946 m/z SELDI-TOF MS peak was found to retain discriminatory value in each of two separate data sets with an increased expression in the diseased state. Sequence identification by liquid chromatography-MS/MS and subsequent immunoassays verified that an isoform of apolipoprotein A-II (ApoA-II) is the observed 8,946 m/z SELDI peak. Immunohistochemistry revealed that ApoA-II is overexpressed in prostate tumors. SELDI-based immunoassay revealed that an 8.9-kDa isoform of ApoA-II is specifically overexpressed in serum from individuals with prostate cancer. ApoA-II was also overexpressed in the serum of individuals with prostate cancer who have normal prostate-specific antigen (0-4.0 ng/mL). CONCLUSIONS: We have identified an isoform of ApoA-II giving rise to an 8.9K m/z SELDI "peak" that is specifically overexpressed in prostate disease. The ability of ApoA-II to detect disease in patients with normal prostate-specific antigen suggests potential utility of the marker in identifying indolent disease.     

Trans-perineal implantation of radio-opaque treatment verification markers into the prostate: an assessment of procedure related morbidity, patient acceptability and accuracy.

On-line imaging of prostate markers can be used to compensate for errors in radiation delivery. This study assessed the patient acceptability and morbidity associated with the trans-perineal route of implantation. A minority experienced acute pain or bleeding. Placement was accurate in all but one subject. An operator related learning curve exists. Although this is an invasive procedure most patients found it acceptable. Implementation for routine clinical practice is feasible.     

Androgen receptors beyond prostate cancer: an old marker as a new target

Androgen receptors (ARs) play a critical role in the development of prostate cancer. Targeting ARs results in important salutary effects in this malignancy. Despite mounting evidence that ARs also participate in the pathogenesis and/or progression of diverse tumors, exploring the impact of hormonal manipulation of these receptors has not been widely pursued beyond prostate cancer. This review describes patterns of AR expression in a spectrum of cancers, and the potential to exploit this knowledge in the clinical therapeutic setting.     

Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer.

PURPOSE: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps due to occult metastatic disease. One potential solution is the utilization of a well-tolerated systemic therapy (e.g., vaccine) in concert with local therapy. EXPERIMENTAL DESIGN: We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapy-only arms. Those patients in the combination arm received a "priming" vaccine with recombinant vaccinia (rV) PSA plus r V containing the T-cell costimulatory molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local granulocyte-macrophage colony-stimulating factor and low-dose systemic interleukin-2. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations. RESULTS: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases in PSA-specific T cells of at least 3-fold versus no detectable increases in the radiotherapy-only arm (P < 0.0005). There was also evidence of de novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence of immune-mediated tumor killing. The vaccine was well tolerated. CONCLUSION: This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the majority of patients generating a PSA-specific cellular immune response to vaccine.     

Cell proliferation in prostate cancer patients with lymph node metastasis: a marker for progression.

The biological aggressiveness of lymph node-positive prostate cancer is closely linked to cancer volume in nodal metastases. We evaluated MIB-1 (Ki-67) labeling index and bcl-2 expression in primary cancer and matched nodal metastases from 138 node-positive patients treated with radical prostatectomy and bilateral pelvic lymphadenectomy between 1987 and 1992 at the Mayo Clinic. One hundred twenty-eight patients (93%) received androgen deprivation therapy within 90 days after radical prostatectomy. Mean patient age was 66 years (range, 51-78). The median follow-up was 6.7 years (range, 0.03-11). MIB-1 (Ki-67) labeling index was determined by digital image analysis, and nodal cancer volume was determined by the grid method. Systemic progression, defined as the presence of distant metastasis documented by biopsy or radiographic examination, was used as an outcome end point in the Cox proportional hazard models. MIB-1 labeling index in nodal metastases was predictive of systemic progression-free survival (P = 0.001). The 8-year systemic progression-free survival was 100% for those with MIB-1 labeling index <3.5% compared with 78% for those with MIB-1 labeling index > or =7.8%. MIB-1 labeling index correlated with Gleason score, DNA ploidy, and nodal cancer volume (P<0.001, 0.04, and <0.001, respectively). After controlling for nodal cancer volume, MIB-1 labeling index remained significant in predicting systemic progression-free survival (P = 0.047). bcl-2 expression in the primary cancer and lymph node metastasis was associated with systemic progression-free survival in univariate analysis (P = 0.027 and 0.048, respectively) but was not significant after adjusting for nodal cancer volume (P = 0.52 and 0.17, respectively). Our data indicate that assessment of cell proliferation in nodal metastasis is predictive of clinical outcome in prostate cancer patients with regional lymph node metastasis.     

Comparison of daily megavoltage electronic portal imaging or kilovoltage imaging with marker seeds to ultrasound imaging or skin marks for prostate localization and treatment positioning in patients with prostate cancer

PURPOSE: To compare the accuracy of imaging modalities, immobilization, localization, and positioning techniques in patients with prostate cancer. METHODS AND MATERIALS: Thirty-five patients with prostate cancer had gold marker seeds implanted transrectally and were treated with fractionated radiotherapy. Twenty of the 35 patients had limited immobilization; the remaining had a vacuum-based immobilization. Patient positioning consisted of alignment with lasers to skin marks, ultrasound or kilovoltage X-ray imaging, optical guidance using infrared reflectors, and megavoltage electronic portal imaging (EPI). The variance of each positioning technique was compared to the patient position determined from the pretreatment EPI. RESULTS: With limited immobilization, the average difference between the skin marks' laser position and EPI pretreatment position is 9.1 +/- 5.3 mm, the average difference between the skin marks' infrared position and EPI pretreatment position is 11.8 +/- 7.2 mm, the average difference between the ultrasound position and EPI pretreatment position is 7.0 +/- 4.6 mm, the average difference between kV imaging and EPI pretreatment position is 3.5 +/- 3.1 mm, and the average intrafraction movement during treatment is 3.4 +/- 2.7 mm. For the patients with the vacuum-style immobilization, the average difference between the skin marks' laser position and EPI pretreatment position is 10.7 +/- 4.6 mm, the average difference between kV imaging and EPI pretreatment position is 1.9 +/- 1.5 mm, and the average intrafraction movement during treatment is 2.1 +/- 1.5 mm. CONCLUSIONS: Compared with use of skin marks, ultrasound imaging for positioning provides an increased degree of agreement to EPI-based positioning, though not as favorable as kV imaging fiducial seeds. Intrafraction movement during treatment decreases with improved immobilization.     

Reg IV: a promising marker of hormone refractory metastatic prostate cancer.

The diagnosis and management of prostate cancer is hampered by the absence of markers capable of identifying patients with metastatic disease. In order to identify potential new markers for prostate cancer, we compared gene expression signatures of matched androgen-dependent and hormone refractory prostate cancer xenografts. One candidate gene overexpressed in a hormone refractory xenograft was homologous to the regenerating protein gene family, a group of secreted proteins expressed in the gastrointestinal tract and overexpressed in inflammatory bowel disease and cancer. This gene, Reg IV, was confirmed to be differentially expressed in the LAPC-9 hormone refractory xenograft. Consistent with its up-regulation in a hormone refractory xenograft, it is expressed in several prostate tumors after neoadjuvant hormone ablation therapy. As predicted by its sequence homology, it is secreted from transiently transfected cells. It is also expressed strongly in a majority of hormone refractory metastases represented on two high-density tissue microarrays. In comparison, it is not expressed by any normal prostate specimens and only at low levels in approximately 40% of primary tumors. These data support Reg IV as a candidate marker for hormone refractory metastatic prostate cancer.     

Immunohistochemical detection of a hypoxia marker in spontaneous canine tumours.

An immunoperoxidase technique has been used to detect the in vivo binding of a 2-nitroimidazole hypoxia marker in histochemical sections of a variety of excised canine tumours. The binding occurred 10-12 cell diameters away from tumour blood vessels, consistent with the expected location of hypoxic cells in tissues in which oxygen concentration gradients are established by diffusion. Hypoxic fractions ranging from 4 to 13% have been estimated on the basis of morphometric analysis of multiple tumour sections. The binding of the marker was restricted to the cytoplasm of the cells. The marker appeared in regions adjacent to necrosis but also in regions free of necrosis. As in earlier autoradiography studies, binding was occasionally observed in cells adjacent to tumour blood vessels. Generally, binding to normal tissues was not observed. However, binding to smooth muscle cells surrounding arterioles in some sections of normal tissue and tumour tissue was observed.     

Use of an implanted marker and real-time tracking of the marker for the positioning of prostate and bladder cancers

Purpose: A real-time tracking radiotherapy was investigated to assess its usefulness in precise localization and verification of prostate and bladder cancers.

Methods and Materials: The real-time tracking radiation therapy (RTRT) system consists of implantation of a 2.0-mm gold marker into a clinical target volume (CTV), three-dimensional radiation treatment planning (3DRTP) system, and the use of two sets of diagnostic x-ray television systems in the linear accelerator room, image processing units, and an image display unit. The position of the patient can be corrected by adjusting the actual marker position to the planned marker position, which has been transferred from the 3DRTP and superimposed on the fluoroscopic image on the display unit of the RTRT system. The position of the markers can be visualized during irradiation and after treatment delivery to verify the accuracy of the localization. Ten patients with prostate cancer and 5 patients with bladder cancer were examined using this system for the treatment setup on 91 occasions.

Results: After manual setup using skin markers, the median of absolute value of discrepancies between the actual position of the marker and the planned position of the marker for prostate cancer was 3.4 (0.1–8.9) mm, 4.1 (0.2–18.1) mm, and 2.3 (0.0–10.6) mm for the lateral, anteroposterior, and craniocaudal directions, respectively. The 3D median distance between the actual and planned positions of the marker was 6.9 (1.1–18.2) mm for prostate cancer and 6.9 (1.7–18.6) mm for bladder cancer. After relocation using RTRT, the 3D distance between the actual and planned position of the marker was 0.9 ± 0.9 mm. Median 3D distances between actual positions after treatment delivery and planned positions were 1.6 (0.0–6.3) mm and 2.0 (0.5–8.0) mm during daily radiotherapy for the marker in patients with prostate cancer and bladder cancer, respectively.

Conclusion: We believe the new positioning system can reduce uncertainty due to setup error and internal organ motion, although further improvement is needed for the system to account for the rotational and elastic changes of the affected tissues.     

Implementation and utility of a daily ultrasound-based localization system with intensity-modulated radiotherapy for prostate cancer

: To evaluate the clinical feasibility of daily computer-assisted transabdominal ultrasonography for target position verification in the setting of intensity-modulated radiotherapy (IMRT) for prostate cancer.

: Twenty-three patients with clinically localized prostate cancer were treated using a sequential tomotherapy IMRT technique (Peacock) and daily computer-assisted transabdominal ultrasonography (BAT) for target localization. Patients were instructed to maintain a full bladder and were placed in the supine position using triangulation tattoos and a leg immobilizer to minimize pelvic rotation. The BAT ultrasound system is docked to the treatment collimator and electronically imports the CT simulation target contours and isocenter. The system is able to use the machine isocenter as a reference point to overlay the corresponding CT contours onto the ultrasound images captured in the transverse and sagittal planes. A touch screen menu is used to maneuver the CT contours in three dimensions such that they match the ultrasound images. The system then displays the three-dimensional couch shifts required to produce field alignment. Data were prospectively collected to measure the frequency by which useful ultrasound images were obtained, the amount of time required for localization/setup, and the direction/magnitude of the positional adjustments.

: Of the 23 patients, the BAT ultrasound system produced images of sufficient quality to perform the overlay of the CT contours in 19 patients such that positional verification could be reliably performed. Poor image quality was associated with patient inability to maintain a full bladder, large body habitus, or other anatomic constraints. Of the 19 assessable patients, a total of 185 treatment alignments were performed (mean 8.8/patient). For all cases, the average time required for the daily ultrasound imaging and positional adjustments was 11.9 min. After the initial 5 cases, the user experience skills improved such that the time required for image verification/positional adjustments decreased to a mean of 5.6 min. The average right-left, AP, and cranial-caudal adjustment was 2.6 ± 2.1 mm, 4.7 ± 2.7 mm, and 4.2 ± 2.8 mm, respectively. Positional adjustments >10 mm were infrequent and related primarily to misidentification of the target structures on the ultrasound image, patient movement, or improper registration of the triangulation tattoos.

: Daily computer-assisted BAT ultrasound positional verification of the prostate can be successfully performed through the acquisition of high-quality images in most patients with only a modest increase in treatment setup time. Positional data obtained with this system resulted in clinically meaningful adjustments in daily setup for sequential IMRT that would not be otherwise apparent from other verification modalities.     

Prostate-specific antigen. Monitoring the response of carcinoma of the prostate to radiotherapy with a new tumor marker.

The role of prostate-specific antigen (PSA), a sensitive tumor marker for cancer of the prostate, has yet to be defined in patients treated with radiotherapy. To evaluate this, PSA and acid phosphatase (AP) were measured prospectively in 110 sequential patients who presented with locoregional carcinoma of the prostate and in whom radiotherapy was to be definitive treatment. Therapy was divided into the following treatment groups: external-beam radiotherapy alone (EBRT), EBRT with brachytherapy (EBRT + B), and hormone therapy either pre-EBRT or post-EBRT (EBRT + H). All patients have been followed for 1 to 17 months and a total of 521 posttreatment PSA determinations have been made. In 91 of 110 patients (83%) PSA was elevated pretreatment and correlated with clinical stage and subsequent relapse. There was no association with Gleason grade, assigned treatment group, or lymph node involvement. Acid phosphatase was elevated in only 31% of the patients initially and had no predictive value in subsequent failure. Nine patients have developed local and/or distant recurrence. None of the patients who failed had their PSA return to normal whereas 74 of 101 (73%) of the remainder have done so. Levels of PSA that do not return to normal during follow-up probably indicate active disease, often without evidence of clinical relapse. The authors conclude that PSA is a useful tumor marker for monitoring response to radiotherapy and may be a predictor of eventual failure thus identifying patients eligible for early intervention therapy as and when it becomes available.     

Stereographic targeting in prostate radiotherapy: speed and precision by daily automatic positioning corrections using kilovoltage/megavoltage image pairs

PURPOSE: A fully automated, fast, on-line prostate repositioning scheme using implanted markers, kilovoltage/megavoltage imaging, and remote couch movements has been developed and clinically applied. The initial clinical results of this stereographic targeting (SGT) method, as well as phantom evaluations, are presented. METHODS AND MATERIALS: Using the SGT method, portal megavoltage images are acquired with the first two to six monitor units of a treatment beam, immediately followed by acquisition of an orthogonal kilovoltage image without gantry motion. The image pair is automatically analyzed to obtain the marker positions and three-dimensional prostate displacement and rotation. Remote control couch shifts are applied to correct for the displacement. The SGT performance was measured using both phantom images and images from 10 prostate cancer patients treated using SGT. RESULTS: With phantom measurements, the accuracy of SGT was 0.5, 0.2, and 0.3 mm (standard deviation [SD]) for the left-right, craniocaudal, and anteroposterior directions, respectively, for translations and 0.5 degrees (SD) for the rotations around all axes. Clinically, the success rate for automatic marker detection was 99.5%, and the accuracy was 0.3, 0.5 and 0.8 mm (SD) in the left-right, craniocaudal, and anteroposterior axes. The SDs of the systematic center-of-mass positioning errors (Sigma) were reduced from 4.0 mm to <0.5 mm for all axes. The corresponding SD of the random (sigma) errors was reduced from 3.0 to <0.8 mm. These small residual errors were achieved with a treatment time extension of <1 min. CONCLUSION: Stereographic targeting yields systematic and random prostate positioning errors of <1 mm with <1 min of added treatment time.     

microRNA-183 is an oncogene targeting Dkk-3 and SMAD4 in prostate cancer

Background:

The purpose of this study was to identify prostate cancer (PC) oncogenic microRNAs (miRs) based on miR microarray and to investigate whether these oncogenic miRs may be useful as PC biomarkers.

Methods:

Initially, we carried out miR microarray and real-time PCR using RWPE-1, PC-3, DU-145 and LNCaP cells. To investigate the function of miR-183, we used a miR-183 knockdown inhibitor in cell growth and wound-healing assays. We used several algorithms and confirmed that they are directly regulated by miR-183.

Results:

We identified three potential oncogenic miRs (miR-146a, miR-183 and miR-767-5P). The expression of miR-183 in PC cells (PC-3, DU-145 and LNCaP) was upregulated compared with RWPE-1 cells. MiR-183 expression was also significantly higher in PC tissues compared with that in matched normal prostate tissues. Additionally, miR-183 expression was correlated with higher prostate-specific antigen, higher pT and shorter overall survival. MiR-183 knockdown decreased cell growth and motility in PC cells and significantly decreased prostate tumour growth in in vivo nude mice experiments. We identified Dkk-3 and SMAD4 as potential target genes of miR-183.

Conclusion:

Our data suggest that oncogenic miR-183 may be useful as a new PC biomarker and that inhibition of miR-183 expression may be therapeutically beneficial as a PC treatment.     

RASSF1A is not appropriate as an early detection marker or a prognostic marker for non-small cell lung cancer

Aberrant methylation of several tumor suppressor genes often occurs during the pathogenesis of lung cancer. RASSF1A is one of the tumor suppressor genes, and it is frequently inactivated by hypermethylation of its promoter region in a variety of human cancers, including lung cancer. It has recently been suggested that RASSF1A methylation was frequently observed in poorly differentiated tumors, and that it was correlated with adverse survival in lung adenocarcinoma (Tomizawa Y, et al., Clin Cancer Res 2002;8:2362-8). In this study, we investigated the pathogenetic and clinicopathologic significance of RASSF1A methylation for the development and/or progression of non small cell lung cancer (NSCLC). We examined 116 cases of NSCLC for the methylation status of RASSF1A. Methylation-specific analysis demonstrated that 40.5% (47 of 116) of the cases were methylated at the CpG sites in the promoter. Methylation of RASSF1A was associated with cellular differentiation (p = 0.0244) and it was related to survival (p = 0.0276). However, there was no association between RASSF1A methylation and the individual clinicopathologic features: TNM stage (p > 0.1), recurrence (p > 0.1), lymphatic permeation (p > 0.1) and smoking duration time (p > 0.1). Furthermore, we analyzed RASSF1A's probability as a prognostic marker by using stepwise Cox proportional hazard regression testing. As a result, the stage proved to be the most important factor (p = 0.0089), more than any other factors such as age, gender, cell type, methylation status, differentiation, smoking duration time, tumor size and lymph node permeation. There was no other significant factor other than stage and age. These results show that epigenetic inactivation of RASSF1A cannot be a prognostic marker of NSCLC.     

Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men

The prostate-specific antigen test has been a major factor in increasing awareness and better patient management of prostate cancer (PCA), but its lack of specificity limits its use in diagnosis and makes for poor early detection of PCA. The objective of our studies is to identify better biomarkers for early detection of PCA using protein profiling technologies that can simultaneously resolve and analyze multiple proteins. Evaluating multiple proteins will be essential to establishing signature proteomic patterns that distinguish cancer from noncancer as well as identify all genetic subtypes of the cancer and their biological activity. In this study, we used a protein biochip surface enhanced laser desorption/ionization mass spectrometry approach coupled with an artificial intelligence learning algorithm to differentiate PCA from noncancer cohorts. Surface enhanced laser desorption/ionization mass spectrometry protein profiles of serum from 167 PCA patients, 77 patients with benign prostate hyperplasia, and 82 age-matched unaffected healthy men were used to train and develop a decision tree classification algorithm that used a nine-protein mass pattern that correctly classified 96% of the samples. A blinded test set, separated from the training set by a stratified random sampling before the analysis, was used to determine the sensitivity and specificity of the classification system. A sensitivity of 83%, a specificity of 97%, and a positive predictive value of 96% for the study population and 91% for the general population were obtained when comparing the PCA versus noncancer (benign prostate hyperplasia/healthy men) groups. This high-throughput proteomic classification system will provide a highly accurate and innovative approach for the early detection/diagnosis of PCA.     

Establishment of a serum tumor marker for preclinical trials of mouse prostate cancer models.

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.