Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease. METHODS: We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa. RESULTS: We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an 'interactome' with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6. CONCLUSIONS: Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Identification of markers of prostate cancer progression using candidate gene expression.

BACKGROUND: Metastatic prostate cancer (PCa) has no curative treatment options. Some forms of PCa are indolent and slow growing, while others metastasise quickly and may prove fatal within a very short time. The basis of this variable prognosis is poorly understood, despite considerable research. The aim of this study was to identify markers associated with the progression of PCa. METHODS: Artificial neuronal network analysis combined with data from literature and previous work produced a panel of putative PCa progression markers, which were used in a transcriptomic analysis of 29 radical prostatectomy samples and correlated with clinical outcome. RESULTS: Statistical analysis yielded seven putative markers of PCa progression, ANPEP, ABL1, PSCA, EFNA1, HSPB1, INMT and TRIP13. Two data transformation methods were utilised with only markers that were significant in both selected for further analysis. ANPEP and EFNA1 were significantly correlated with Gleason score. Models of progression co-utilising markers ANPEP and ABL1 or ANPEP and PSCA had the ability to correctly predict indolent or aggressive disease, based on Gleason score, in 89.7% and 86.2% of cases, respectively. Another model of TRIP13 expression in combination with preoperative PSA level and Gleason score was able to correctly predict recurrence in 85.7% of cases. CONCLUSION: This proof of principle study demonstrates a novel association of carcinogenic and tumourigenic gene expression with PCa stage and prognosis.

Pharmacokinetics of once-daily gentamicin dosing in pediatric patients.

BACKGROUND/PURPOSE: To achieve cost-effective health care in adults, once-daily aminoglycosides administration has been used and judged to be safe and efficacious. A similar strategy in children requires the characterization of pharmacokinetic parameters and the development of a therapeutic monitoring protocol for this antibiotic regimen. METHODS: A prospective, controlled, randomized (2:1) study was undertaken in 50 pediatric patients between June 1995 and September 1997. Children between 6 months and 18 years who required gentamicin therapy based on independent clinical assessment were eligible if they had normal renal function, no aminoglycoside allergies, were not neutropenic, or did not have cystic fibrosis. Measurements included a peak, 4-hour, 8-hour, and trough gentamicin levels to determine volume of distribution (Vd) and elimination constant (Ke). Ototoxicity and nephrotoxicity were monitored by pre- and postaudiology examinations and serial calculated creatinine clearance determinations, respectively. RESULTS: Thirty-three patients received 7.5 mg/kg every 24 hours, and 17 patients received 2.5 mg/kg every 8 hours. Most frequent indications for treatment were ruptured appendicitis (n = 19) followed by wound infections caused by trauma (n = 4), but the spectrum of treatment was broad including enteric, genitourinary, central nervous system, biliary, ophthalmologic, and orthopedic infections. Pharmacokinetic data indicated that 24-hour dosing resulted in higher peak levels compared with 8-hour dosing (20.4 +/- 45.4 v 7.2 +/- 6.2 mg/L, P < .0001) and lower trough levels (0.29 +/- .02 v 0.69 +/- 0.13, P < .0001), whereas rate of elimination constant and volume of distribution were not significantly different. No nephrotoxicity or ototoxicity has been noted in either group. CONCLUSIONS: These data confirm that once-daily dosing of gentamicin is a safe method of treatment that provides equivalent pharmacokinetics compared with traditional dosing and enhances bactericidal effect based on higher peak levels, avoids toxicity, and allows cost savings.

Epithelial markers in pancreatic carcinoma: immunoperoxidase localisation of DD9, CEA, EMA and CAM 5.2.

Paraffin wax embedded, formalin fixed sections of 22 adenocarcinomas of the exocrine pancreas were stained with four mouse monoclonal antibodies: DD9-E7, an antibody raised against a human pancreatic tumour xenograft; carcino-embryonic antigen (CEA); epithelial membrane antigen (EMA); and cytokeratin (CAM 5.2). An indirect immunoperoxidase technique without enzyme pre-digestion and an affinity-purified sheep anti-mouse peroxidase conjugate were used. All of the tumours were positive for DD9-E7, EMA, and CAM 5.2. Twenty out of 22 were focally positive for CEA and the staining was often weak. As all of these adenocarcinomas were DD9-E7 positive, absence of staining for DD9-E7 in a tumour makes the diagnosis of adenocarcinoma of the exocrine pancreas very unlikely, and this is of value in distinction from endocrine carcinomas with a marked acinar pattern. The weak CEA staining distinguished pancreatic carcinomas from colorectal tumours. Because the distribution of staining for EMA and CAM 5.2 was no different from that previously seen in adenocarcinomas from other sites, these markers are likely to be of limited value in the differential diagnosis of abdominal adenocarcinomas of uncertain origin.

A novel hybridoma antibody (PASE/4LJ) to human prostatic acid phosphatase suitable for immunohistochemistry.

A murine monoclonal antibody PASE/4LJ to prostatic acid phosphatase (PAP) was used to immunostain a wide variety of sections of benign and malignant tissues (654 blocks). Non-neoplastic adult and fetal prostatic glands, primary and metastatic prostatic carcinomas, and scattered cells in prostatic and penile urethra were positive. Rat, dog and rabbit prostates were negative. Nine of 400 tumours of non-prostatic origin showed some positivity: 6/36 carcinoids, 1/9 islet cell tumours, 1/55 ovarian adenocarcinomas (serous) and one carcinosarcoma of the lung (epithelial portion). Positive staining was seen in islet cells in 4/5 specimens of normal pancreas, and in 4/9 blocks of normal pancreas surrounding a pancreatic tumour. Loops of Henle, maculae densae, and distal tubules in 10/10 fetal and 2/9 adult kidneys were also positive, with proximal tubules and collecting ducts negative. All other 159 blocks of non-neoplastic adult and fetal tissues were negative. The antibody was also affinity purified from ascitic fluid, and shown not to inhibit the enzyme activity of prostatic acid phosphatase.

Response of breast carcinoma to endocrine therapy predicted using immunostained pelleted fine needle aspirates.

Fine needle aspirates from 82 patients with breast carcinoma were fixed in methacarn, double embedded in agar or gelatin, and then in paraffin wax. Sequential sections were stained with monoclonal antibodies to the oestrogen receptor-related protein P29 (antibody D5), carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and cytokeratin (CAM 5.2). Sixty-one of 82 (74%) aspirates provided sections suitable for immunostaining. Twenty-six (43%) were D5 positive, 23 (38%) CEA positive, 59 (97%) EMA positive, and 54 (89%) CAM 5.2 positive. Twenty-six of these patients were treated with some form of endocrine therapy. Twelve (46%) showed positive staining for D5. Eleven (92%) of the 12 D5-positive patients responded or had static disease, and 8% progressed. Of the 14 D5-negative tumours 43% responded or remained static, and 57% progressed. The difference in response between the D5-positive and the D5-negative tumours was significant (P less than 0.05, Fisher's exact test). There was no correlation between staining for CEA, EMA or cytokeratin and response to endocrine therapy.

The generation of monoclonal antibodies against human pancreatic exocrine cancer: a study of six different immunisation regimes.

Six different immunisation regimes have been used to generate spleen cells with reactivity against human pancreatic exocrine cancer. Immunised spleen cells were fused with an NSO/1 myeloma line and supernatants from these hybridomas selectively screened for monoclonal antibodies which bound predominantly to a pancreatic cancer cell line (GER). The spleen cells from hairy litter mates immunised with pancreatic cancer xenograft homogenates and viable GER cells generated 13% of hybridoma supernatants which showed some selectivity for GER pancreatic cancer cells in a fixed cell ELISA assay. The other methods produced only 4% of hybrids with selectivity for GER cells. The antigen distribution on gluteraldehyde fixed cells was similar to that found for viable cell monolayers but many antigens were unstable on formalin fixation. Immunohistochemical staining of GER cells grown on glass slides showed a heterogeneity of antigen distribution with up to 70% of the cells exhibiting a vesicular pattern of staining. Fifty percent of the antibodies which bound to GER cells were also reactive against antigens in formalin-fixed paraffin-embedded tissue sections of the original GER tumour. Monoclonal antibody DD9E7 identified an antigen expressed on 12/14 pancreatic adenocarcinomas. The antibody showed strong staining of malignant luminal membranes and cytoplasm. The antigen was also present in normal salivary and sweat glands, and colon and breast carcinomas, but its tissue distribution was unlike that of CEA or EMA. The expression of this antigen in 12/14 of pancreatic carcinomas suggests that DD9E7 may be a useful reagent for pancreatic tumour detection.