The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/ß-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis.

Molecular detection of Mycobacterium tuberculosis: impact on patient care.

BACKGROUND: Nucleic acid amplification technologies such as PCR are revolutionizing the detection of infectious pathogens such as tuberculosis (TB). Amplification technology offers the potential for the diagnosis of TB in a few hours with a high degree of sensitivity and specificity. However, molecular assays neither replace nor reduce the need for conventional smear and culture, speciation, and antibiotic sensitivity assays. METHODS: We undertook prospective studies of sputum samples to assess the performance of two PCR-based assays for the detection of TB as well as the impact of more rapid availability of test results on patient care. RESULTS: The sensitivity of both the in-house and Amplicor PCR assays was 100% for smear-positive sputa. For smear-negative sputa (two sputum samples collected during the first 24 h of hospitalization), the sensitivity was 85% for our in-house PCR assay and 74% for the Roche PCR assay. Approximately 10% of the smear- and culture-negative sputa yielded positive PCR results; however, more than one-half of these were positive with both the in-house and Amplicor assays, suggesting the presence of TB DNA or organisms. Several of these came from patients whose other samples grew Mycobacterium tuberculosis during the same admission, and others came from patients who had previously treated TB. Overall, the specificities of the in-house and Amplicor PCR assays in smear-negative patients were 86% and 93%, respectively. CONCLUSIONS: Molecular detection of slow-growing pathogens such as M. tuberculosis have the potential to improve clinical care through a dramatic reduction in the time required for detection and may provide substantial savings in the overall cost of care of a patient compared with conventional smear, culture, and speciation alone, despite the fact that conventional assays must still be performed for speciation of nontuberculous mycobacteria and for full assessment of antibiotic sensitivity.

Tumor-infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma.

BACKGROUND: Cells with deficient DNA mismatch repair develop microsatellite instability. Extensive microsatellite instability (MSI-high) is characteristic of colorectal carcinomas in hereditary nonpolyposis colorectal carcinoma (HNPCC) and in 10-% 15% of sporadic colorectal carcinomas. Microsatellite instability-high colorectal carcinomas differ from others in important clinical and pathologic features. However, MSI typing is expensive and not widely available. Microsatellite instability type may be predicted by tumor-infiltrating lymphocytes (TILs), which can be evaluated with ordinary light microscopy. METHODS: The authors evaluated TILs as a pathology screen for MSI-high status in 138 colorectal carcinomas that had been evaluated for MSI in a variety of studies. This case series was systematically enriched with HNPCC and other MSI-high cases to allow accurate sensitivity and specificity estimation. Tumor-infiltrating lymphocytes were quantitated as TILs per 10 high-power microscopic fields by an observer blinded to MSI status. RESULTS: Of the 138 carcinomas studied, 67 (48.6%) were MSI-high, 22 (15.9%) were MSI-low, and 49 (35.5%) were MSI-stable. All 25 HNPCC colorectal carcinomas were MSI-high. Tumor-infiltrating lymphocytes counts ranged from 0 to 300, with a markedly skewed distribution (median, 11; mean, 36). Sensitivity and specificity for selected cut points of TIL count were computed. Using a TIL count of 5 as a cut point yields a sensitivity of 93% and specificity of 62%. In a population in which 12% were MSI-high, consideration of TIL could reduce the number of colorectal carcinomas referred for MSI testing by greater than one-half, and still 93% of the MSI-high carcinomas would be identified. CONCLUSIONS: The presence of MSI defines a subset of colorectal carcinomas with special molecular etiology and characteristic clinical, pathologic features, inclusive of increased survival. The authors conclude that quantification of TILs may provide a simple, single criterion for choosing which colorectal carcinomas are candidates for MSI testing.

Detection of breast cancer cells using immunomagnetic beads and reverse transcriptase polymerase chain reaction.

Molecular methods permit the detection of cells too few in number to be detected by light microscopy, immunohistochemistry, or flow cytometry (1-5). Numerous investigators are therefore developing sensitive and specific reverse transcriptase polymerase chain reaction (RT-PCR) assays for tumor cell detection. The detection of small numbers of tumor cells in blood, lymph node, and stem cell harvests may have a significant impact on our understanding of the spread of breast cancer, and eventually may impact the management of breast cancer patients as well.

Molecular detection of circulating prostate cancer cells.

Molecular methods have proven extremely useful for the detection of occult tumor cells and can yield valuable clinical information as well as a better understanding of the mechanisms of metastasis and relapse of cancer (1). In the case of many hematopoietic malignancies, the presence of a unique molecular marker such as a chromosomal translocation has made this task relatively straightforward. Carcinomas generally lack such markers, however. Certain oncogene mutations have been targeted, but the lack of consistent and specific markers has remained problematic.

Gastric stromal tumor.

Gastric stromal tumors display a bewildering array of immunohistological and ultrastructural features as well as variable biological behavior. These tumors are rare as compared with ones that arise from the gastric epithelium. Moreover, they have been the subjects of controversy because of their uncertain histogenesis. We report the pathological features of gastric stromal tumors we recently encountered in three patients.

A method for the quantitative analysis of human heat shock gene expression using a multiplex RT-PCR assay.

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP) and molecular chaperones hsp90alpha, hsp90beta, hsp70, hsc70, mtHsp75, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcription of total RNA isolated from human cells followed by amplification with PCR. By the careful selection of pairs of oligonucleotide primers corresponding to unique regions of each heat shock gene, selectivity can be attained such that messenger RNAs of multiple heat shock genes can be analyzed simultaneously in a single reaction. This method provides both the absolute and relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.

Clinical and molecular followup after radical retropubic prostatectomy.

PURPOSE: We previously reported evidence of hematogenous dissemination of prostate cells during radical retropubic prostatectomy, and we now provide clinical and molecular reverse transcriptase-polymerase chain reaction (RT-PCR) followup of that patient cohort. MATERIALS AND METHODS: A total of 101 men with clinically localized prostate cancer were prospectively enrolled in the study. The prostate specific antigen (PSA) RT-PCR assay was performed on peripheral venous blood samples preoperatively in 101, during surgery in 29, during and up to 12 weeks after surgery in 50 and at least 1 year postoperatively in 65 patients. Correlation with clinical (PSA) indicators of recurrence was performed. RESULTS: Of the 101 patients 9 demonstrated biochemical evidence of prostate cancer progression (median followup 22 months). Of the 50 men with perioperative molecular results the RT-PCR positive rate increased from 22% preoperatively in 11 to 48% in 24 (p = 0.02) and then decreased to 10% in 4 of 40 men at 1 year postoperatively (p = 0.07). Molecular followup at a minimum of 1 year after radical retropubic prostatectomy was obtained in 65 men, of whom the RT-PCR positive rate decreased from 23% preoperatively in 14 to 9.2% in 6 (p = 0.05). No significant correlation was observed between a persistently positive RT-PCR result and biochemical failure. CONCLUSIONS: Although a significant proportion of men have molecular evidence of hematogenous prostate cell dissemination intraoperatively, longitudinal molecular and clinical followup demonstrates reconversion to a negative status as the predominant trend. At relatively short followup no significant correlation was identified between the RT-PCR result and the PSA progression-free survival.

A new polymerase chain reaction-based method for the detection of T-cell clonality in patients with possible cutaneous T-cell lymphoma.

OBJECTIVE: To evaluate a new, rapid, and sensitive method for the detection of T-cell clonality in patients with lesions suggestive of cutaneous T-cell lymphoma (CTCL). DESIGNS: Skin specimens were obtained from 48 patients with possible CTCL. Polymerase chain reaction amplification of the T-cell receptor gamma (TCRG) gene was performed using consensus primers to the V and J regions. Clonal populations having identical N-region sequences are detected by single-strand conformation polymorphism analysis using a semiautomated electrophoresis system with a silver-staining method for gel visualization. The results of clinicopathological assessment were compared with those of immunohistochemistry and polymerase chain reaction analysis. SETTING: Cutaneous lymphoma clinic at a university medical center. PATIENTS: Forty-eight patients with skin lesions suggestive of CTCL. RESULTS: Based on the clinicopathological assessment, 26 patients were diagnosed as having CTCL. Of them, clonality was detected in 19 patients (73%) and an abnormal phenotype in 17 (68%) of 25 patients. Combining both tests, abnormal results were noted in 24 (92%) of 26 patients with CTCL. Clonality was also identified in 2 (12%) of 17 patients with presumably benign lesions on clinicopathological assessment. CONCLUSIONS: Polymerase chain reaction and single-strand conformation polymorphism analysis of the TCRG gene is a rapid and sensitive method that can contribute to the diagnosis of CTCL. The new method is especially useful when used in conjunction with immunophenotyping.

Diagnosis of pulmonary tuberculosis using PCR assays on sputum collected within 24 hours of hospital admission.

There have been few studies evaluating the efficacy of polymerase chain reaction (PCR) testing in front-line clinical practice. We assessed the diagnostic yield of PCR prospectively in a blinded study of patients admitted to rule out tuberculosis and compared PCR results to a culture and clinical diagnosis of tuberculosis. Specimens were sent for routine smear, culture, and PCR analysis. Sputum sediments were submitted for PCR amplification of IS6110 sequences by an in-house assay and also the Roche Amplicor PCR assay targeting 16s ribosomal RNA genes. Eighty-five patients were enrolled: 27 patients had cultures positive for tuberculosis; 12 were smear-positive. PCR by both assays on the first specimen picked up all patients smear-positive on any specimen. A positive PCR on at least one of two specimens collected in the first 24 h was 85 and 74% sensitive and 88 and 93% specific for tuberculosis by the in-house and Roche techniques, respectively. Sensitivity in smear-negative patients was 73 and 53%, respectively. The in-house PCR detected 100% and Roche detected 95% of patients with more than paucibacillary (greater than 20 colonies) tuberculosis. We conclude that PCR may be a useful tool to evaluate patients for tuberculosis within the first hospital day.

Detection of Breast Cancer Cells in Blood Using Immunomagnetic Bead Selection and Reverse Transcription-Polymerase Chain Reaction.

Background: A sensitive and specific method for the detection of occult breast cancer cells may prove clinically useful. The purpose of this study was to investi gate cytokeratin-19 (CK-19) and gross cystic disease fluid protein (GCDFP) as potential reverse transcription-polymerase chain reaction (RT-PCR) targets for the detection of breast micrometastases. Through positive selection of breast epithelial cells by immunomagnetic bead separation, two RT-PCR assays were developed. Methods and Results: Positive selection of breast epithelial cells was performed using Ber-EP4 monoclonal antibody bound to magnetic beads. RNA was isolated and RT-PCR performed using CK-19 and GCDFP as targets. Detection sensitivity was five T47D cells spiked into 10 mL of normal blood for either target. In all, 100% (3 9/39) of normal bloods were negative for CK-19, whereas only 87% (34/39) were negative with GCDFP. In 15 patients with metastatic disease including 3 without treatment and 12 on either tamoxifen or chemotherapy, CK-19 was positive in 67% (10/15) of patients and GCDFP yielded positives in 27% (4/15) of patients tested. Conclusions: The detection of CK-19 and GCDFP in bloods from patients with metastatic breast cancer may be beneficial in determining the course of therapy, as well as having potential prognostic and diagnostic applications. Although CK-19 appears to be the more sensitive and specific marker, further investigation with both targets is warranted.

Prostate specific antigen reverse transcriptase-polymerase chain reaction assay in preoperative staging of prostate cancer.

PURPOSE: Extracapsular extension of prostate cancer occurs in a significant number of men believed to have clinically localized disease. We report the ability of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay to predict preoperatively the pathological stage of cases of clinically localized prostate cancer. MATERIALS AND METHODS: Since October 1994, 82 consecutive men with clinically localized prostate cancer had a venous blood RT-PCR assessment before radical retropubic prostatectomy. The extracted ribonucleic acid was reverse transcribed, amplified and the amplicon identity confirmed by prostate specific antigen (PSA) directed probe hybridization. An additional 31 patients were enrolled to provide appropriate positive (T + Nx/1M2) and negative (human female and benign prostatic hyperplasia) controls. Histological examination of the entire prostatectomy specimen was performed. RESULTS: Positive RT-PCR assay results correlated significantly with skeletal metastases and elevated levels of serum PSA but they did not significantly improve our ability to identify prospectively patients with extracapsular extension over traditional predictors (serum PSA, Gleason score). CONCLUSIONS: The role of molecular techniques in prostate cancer evaluation and prognosis continues to emerge. However, in our study we demonstrate no significant advantage in preoperative staging of prostate cancer using RT-PCR assay with PSA primers.

Relative expression of epidermal growth factor receptor in placental cytotrophoblasts and choriocarcinoma cell lines.

The role of transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor (EGFR) interactions in regulating benign and malignant trophoblast proliferation were examined. Benign cytotrophoblast (CT) demonstrated mitogenic stimulation in response to TGF-alpha; BeWo and JAr choriocarcinoma cell lines failed to respond. EGFR levels in BeWo and JAr were determined by enzyme linked immunoassay (ELISA) to be at least 10-fold higher than those in benign CT. EGFR isolated from BeWo and JAr also demonstrated functional tyrosine kinase activity. Using a combination of immunoperoxidase (IP) and ELISA techniques, choriocarcinoma cells were found to produce significant quantities of TGF-alpha that were comparable with those reported previously by this laboratory for benign CT, and were felt to be stimulating their own proliferation in an autocrine fashion. EGFR blocking and TGF-alpha neutralizing antibodies inhibited JAr proliferation whereas an EGF neutralizing antibody did not. The data presented here and in our previous report indicate that a TGF-alpha-EGFR autocrine loop may regulate normal and malignant CT proliferation. Choriocarcinoma cells may be proliferating at a maximal rate due, in part, to EGFR overexpression and are therefore unable to respond further to exogenous growth factor. Thus, EGFR overexpression may contribute to the uncontrolled proliferation of choriocarcinoma cells in general.

Mycobacterium tuberculosis DNA Not Detected by Polymerase Chain Reaction In Air Samples From Hospital Rooms of Tuberculosis Patients.

Background: As the incidence of tuberculosis (TB) increased in the United States, the risk of occupational and nosocomial TB also increased. Airborne Mycobacterium tuberculosis (MTB) is difficult to measure directly. Quantifying MTB DNA by polymerase chain reaction in air samples obtained from isolation rooms of patients with TB would provide a measure of the number of airborne organisms produced by a patient and the efficacy of ventilation, and might predict when an individual patient is no longer infectious. Methods and Results: The air was sampled through cellulose ester filters from the isolation room of a patient with newly diagnosed pulmonary TB, from a patient on therapy for 14 days, and from adjacent offices, and an attempt was made to detect MTB DNA; however, MTB DNA was detected only on positive control filters. Conclusions: Mycobacterium tuberculosis was not detected by polymerase chain reaction in air samples from the rooms of two patients with pulmonary TB. This may have been due to the large number of room air changes with resultant rapid clearance of airborne droplet nuclei or to the limited air volume sampled. A sensitive molecular assay of airborne MTB could be used to monitor the efficacy of infection control measures by sampling a sufficient volume of isolation room air and could aid in determining when an individual patient was no longer infectious.