Early tumor cell dissemination in patients with clinically localized carcinoma of the prostate.

Because a significant number of patients with pathologically organ-confined carcinoma of the prostate subsequently develop recurrent disease, metastasis may occur much earlier than previously believed. We have used a reverse transcription-PCR assay for prostate-specific antigen mRNA and an immunocytochemical staining method for cytokeratins to test this hypothesis in paired peripheral blood (PB) and bone marrow (BM) specimens from 71 patients with clinically localized disease before radical prostatectomy, 14 patients with advanced-stage carcinoma of the prostate, and 30 controls (young healthy volunteers, patients without prostate disease, and patients with benign prostatic hyperplasia). Controls were negative in BM and PB. Fifty-six% of patients with organ-confined tumors (pT2) and 73% of those with extracapsular extension (pT3) were positive in the BM versus 16% of those with pT2 tumors and 27% of those with pT3 tumors in the PB. Patients with advanced-stage disease were positive in 86% of BM versus 71% of PB. The sensitivity of the immunocytochemistry assay to detect tumor cells was lower as compared with the reverse transcription-PCR assay. The results suggest that tumor cell dissemination occurs early during disease progression. Prostate cells seem to preferentially concentrate in the BM rather than the PB, which may be due to sequestration there by homing mechanisms. As the rate of detection in the BM exceeds the proportion of patients with subsequently progressing disease, we hypothesize that only a subset of these cells can survive in the BM and evolve to clinically apparent disease.

Decrease in tumor cell contamination and progenitor cell yield in leukapheresis products after consecutive cycles of chemotherapy for breast cancer treatment.

In this retrospective study, we assessed the impact of each of three consecutive cycles of conventional-dose chemotherapy on CD34+ cells, colony-forming units granulocyte-macrophage (CFU-GM), and contaminating breast cancer cells collected in the leukapheresis products of patients with metastatic breast cancer. The patients subsequently underwent high-dose chemotherapy followed by autologous blood progenitor cell transplantation. We analyzed 172 leukapheresis products from 17 patients and have correlated the long-term clinical outcome with tumor cell contamination. The induction chemotherapy regimen consisted of three cycles of cyclophosphamide 750 mg/m2 i.v., epirubicin 100 mg/m2, and 5-fluorouracil (5-FU) 750 mg/m2 i.v., followed by 5 microg/kg body weight of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) daily until leukapheresis was completed. An average of 10 leukapheresis products (three to four collections after each cycle of chemotherapy) were obtained from each patient. Numbers of CD34+ cells, CFU-GM, and mononuclear cells (MNCs) in the leukapheresis products were determined at the time of collection. Aliquots from the same products were frozen and breast cancer cells were detected by immunocytochemistry with a cocktail of anti-cytokeratin antibodies (AE-1, AE-3, CAM 5.2, Keratin 8+18+19) using a standardized immunoalkaline phosphatase method. A minimum of 10(6) cells were examined by light microscopy and by at least two blinded observers. Cells were considered positive when immunostaining was detected in the cytoplasm and on the cell membrane, and cellular morphology was consistent with a malignant phenotype. Of the 172 samples analyzed, 13 of 57 (23%) leukapheresis products collected after cycle I were positive for tumor cells; 3 of 60 (5%) after cycle II; and 4 of 55 (7%) after cycle III. The likelihood of contamination by breast cancer cells after cycle I was significantly higher than after subsequent cycles of chemotherapy (p = 0.0052). Simultaneously, there was a significant decrease in quantity of CD34+ cells and CFU-GM (p < 0.0001 for both comparisons). Our study indicated that leukapheresis products collected after the second or third cycles of induction chemotherapy carry a significantly lower likelihood of tumor cell contamination, albeit the quantity of CD34+ cells or CFU-GM collected was also significantly reduced.

Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a.

Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a. Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis. Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP. The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced. Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis. Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes. Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants. Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products. Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad.

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.