Impact of NADiA ProsVue PSA slope on secondary treatment decisions after radical prostatectomy.

BACKGROUND: Selecting appropriate candidates for postprostatectomy radiotherapy is challenging, because adverse pathological features cannot accurately predict clinical recurrence. Biomarkers that identify residual disease activity may assist clinicians when counseling patients on the risks, benefits and costs of secondary treatment. NADiA ProsVue PSA slope results ≤2.0 pg ml(-1) month(-1) are predictive of a reduced risk of clinical recurrence; however, its clinical utility has not yet been studied. METHODS: We prospectively enrolled men treated by radical prostatectomy in a multicenter, institutional review board-approved clinical trial. At postsurgical follow-up, investigators (N=17) stratified men into low-, intermediate- or high-risk groups for prostate cancer recurrence based on clinicopathological findings and other factors. Investigators documented their initial treatment plan for each subject and serially collected three serum samples for ProsVue testing. After the ProsVue result was reported, investigators recorded whether or not the initial treatment plan was changed. The proportion of cases referred for secondary treatment before and after ProsVue was reported, and the significance of the difference determined. RESULTS: Complete assessments were reported for 225 men, 128 (56.9%) of whom were stratified into intermediate- and high-risk groups. Investigators reported that they would have referred 41/128 (32.0%) at-risk men for secondary treatment. However, after results were known, they referred only 15/128 (11.7%) men. The difference in proportions (-20.3%, 95% confidence interval (CI) -29.9 to -10.3%) is significant (P<0.0001). Odds of a referral was significantly reduced after results were reported (odds ratio 0.28, 95% CI 0.15-0.54, P<0.0001). CONCLUSIONS: Knowledge of a ProsVue result had significant impact on the final treatment plan. A ProsVue result ⩽2.0 pg ml(-1) month(-1) significantly reduced the proportion of men at risk of recurrence who otherwise would have been referred for secondary treatment.

In vivo transduction of cerebellar Purkinje cells using adeno-associated virus vectors.

We investigated whether adenovirus or adeno-associated virus vectors can transduce cerebellar Purkinje cells (PCs) in vivo. Mice were injected in the deep cerebellar nuclei (DCN) with lacZ-transducing adenovirus (Ad.RSV-betagal) or a recombinant AAV serotype 2 (rAAV2) vector (vTR-CMVbeta) mixed with wild-type adenovirus type 5 (Ad5). One week later, Ad.RSV-betagal transduced cells were found throughout the cerebellar white matter in a dose-dependent manner, but few transduced PCs were evident. In contrast, vTR-CMVbeta with Ad5 transduced several hundred PCs throughout the injected hemisphere. Using an rAAV2 vector transducing a CMV-regulated green fluorescent protein gene, we again found PC transduction, but only with Ad5 coinjection. To assess the effect of injection site and to determine whether the apparent requirement for Ad5 coinfection is observed with other promoters, a beta-actin-regulated vector was injected with or without Ad5 to DCN or cerebellar cortical sites. Thousands of transduced PCs were observed under each condition. Cortical injection yielded greater numbers of transduced cells. Injection of rAAV2 without Ad5 led to greater specificity for PC transduction. We conclude that injection of rAAV2 vectors into the cerebellum is an effective means for transferring genes into substantial numbers of Purkinje cells in vivo.

Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity.

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 10(7) FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 x 10(6) and 1 x 10(6) cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells. (Blood. 2000;96:1334-1341)

Maintenance of differential methotrexate toxicity between cells expressing drug-resistant and wild-type dihydrofolate reductase activities in the presence of nucleosides through nucleoside transport inhibition.

Methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR), has been used widely as a chemotherapeutic agent and as a selective agent for cells expressing drug-resistant DHFR activity. MTX deprives rapidly dividing cells of reduced folates that are necessary for thymidylate synthesis and de novo purine nucleotide synthesis. However, MTX toxicity can be circumvented by salvaging thymidine (TdR) and purine nucleosides. Here we have investigated conditions under which nucleoside transport inhibition can be used to maintain differential MTX toxicity between unmodified cells and cells expressing drug-resistant DHFR activity in the presence of exogenous nucleosides. PA317 cells (a 3T3 derivative cell line) were rescued from the toxicity of 0.1 microM MTX by 1.0 microM TdR in the presence of 100 microM inosine. The nucleoside transport inhibitor dipyridamole (DP) resensitized these cells to MTX, even in the presence of exogenous nucleosides. Furthermore, PA317 cells transduced with any of three retroviruses encoding drug-resistant DHFRs remained resistant to MTX over all concentrations tested (up to 10.0 microM) in the presence of DP. Similar results were obtained in transduced HuH7 and K562 cell lines, a human hepatoma and a human leukemia cell line, respectively. We conclude that nucleoside transport inhibition increases the toxicity and selectivity of MTX in cultured cells, and therefore is an effective way to maintain differential MTX toxicity between unmodified and DHFR-modified cells. Our results support the use of nucleoside transport inhibition in in vivo selection protocols involving the liver and hematopoietic systems.

Index, comprehensive microsatellite, and unified linkage maps of human chromosome 14 with cytogenetic tie points and a telomere microsatellite marker.

Three sets of linkage maps (index, comprehensive microsatellite, and unified) have been constructed for human chromosome 14 based on genotypes from the CEPH reference pedigrees. The index maps consist of 18 microsatellite markers, with heterozygosities of at least 68% and intermarker spacing no greater than 11 cM. The sex-average comprehensive microsatellite map is 125 cM in length and includes 115 markers with 54 loci uniquely placed with odds for marker order of at least 1000:1. The sex-average index map length is 121 cM, and the female- and male-specific maps are 143 and 101 cM, respectively. A unified map was also constructed from 147 loci (162 marker systems), which includes 32 RFLP markers in addition to the 115 microsatellites. The sex-average length of the unified map is 128 cM with 69 loci uniquely placed. Our maps are anchored by a microsatellite telomere marker sCAW1 (D14S826), developed from a telomere YAC clone TYAC196, which extends the linkage map to the physical terminus of the long arm of chromosome 14. Furthermore, we have also physically mapped seven of the loci by fluorescence in situ hybridization of cosmid clones or Alu-PCR products amplified from YACs containing the marker sequences. Together with previously established cytogenetic map designations for other loci, our maps display links between genetic markers for 10 of 13 cytogenetic bands of chromosome 14 at the 550 genome band resolution.