Tissue microarray-based study of patients with lymph node-positive breast cancer shows tyrosine phosphorylation of signal transducer and activator of transcription 3 (tyrosine705-STAT3) is a marker of good prognosis

BACKGROUND: Although lymph node-positive breast cancers are associated with poorer prognosis, individual patients may have different clinical outcomes. Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signalling pathways. The goal of this study was to determine the prognostic value of phosphorylated (tyrosine705)-STAT3 in node-positive breast cancer patients. METHODS: Immunohistochemical analysis of Phospho- STAT3 was performed on a tissue microarray of breast cancer specimens. The expression pattern of Phospho-STAT3 was correlated with survival outcome, and clinical and pathological parameters. RESULTS: Out of 125 interpretable tumours, positive Phospho- STAT3 nuclear expression was seen in 35 (28%) of tumours. There was no significant relationship between Phospho-STAT3 expression and clinical-pathological parameters including age, hormonal receptor status, grade and tumour size. Interestingly positive tumours had a significantly improved disease-free survival at 5 years (p=0.035). Additionally, positive Phospho-STAT3 nuclear expression was correlated with significantly improved survival at both 5 years (p=0.023) and 10 years (p=0.026). Finally, in multivariate analyses Phospho-STAT3 was found to be an independent prognostic marker of overall survival in node-positive breast cancer patients. CONCLUSION: These findings support the role of Phospho- STAT3 as an important independent prognostic marker in node-positive breast cancer patients (AU)

Hyper-IL-6 gene therapy reverses fulminant hepatic failure.

Fulminant hepatic failure is a catastrophic condition caused by massive hepatocellular apoptosis and necrosis. Inhibition of hepatocyte apoptosis and the enhancement of the endogenous potential for liver regeneration could potentially form an effective basis for treatment of this condition. In response to injury in the liver, IL-6 mediates the acute-phase response and induces both cytoprotective and mitogenic functions. Hyper-IL-6 is a superagonistic designer cytokine consisting of human IL-6 linked by a flexible peptide chain to the secreted form of the IL-6 receptor. In a mouse model of acute liver failure induced by d-galactosamine administration, a single low dose of a hyper-IL-6-encoding adenoviral vector, in contrast to an adeno-IL-6 vector, maintained liver function, prevented the progression of liver necrosis, and induced liver regeneration, leading to dramatically enhanced survival. Thus, hyper-IL-6 gene therapy may be useful for the treatment of fulminant hepatic failure, which is often fatal even following treatment by transplantation.

A sensitive and versatile bioluminescence bioassay for HIV type 1 based on adenoviral vectors.

The construction and characterization of a versatile bioassay for the quantification of HIV-1 viral infection and HIV-1 Tat protein activity based on recombinant adenoviral vectors carrying an HIV LTR-driven luciferase reporter gene is described. The assay system consists of a set of two adeno-reporter vectors, one of which is responsive to HIV-1 Tat protein activity, and the second of which is not, by virtue of a deletion of the TAR site within the HIV LTR. This configuration of the reporter genes allows one to distinguish Tat-specific activation from Tat-non-specific HIV LTR-mediated gene expression. The adenoviral HIV LTR-mediated luciferase gene expression is highly responsive to Tat and increases linearly with increasing levels of HIV-1 infection, reaching levels of between 3- and 1000-fold induction. The adeno-reporter viruses can be utilized to detect Tat activity and HIV-1 infection in a wide range of cell types, including 293, CEM, HUT-78, Jurkat, and HeLa-derived cell lines. The resulting bioassay is convenient, sensitive, and readily adaptable to automated procedures. These characteristics of the adeno-reporter assay make it a valuable reagent for studies of HIV infection and for analysis of HIV-inhibitory agents.

Liposome-encapsulated DNA-mediated gene transfer and synthesis of human factor IX in mice.

Hemophilia B is an X-chromosome-linked recessive disorder that is caused by a deficiency of biologically active clotting factor IX (FIX). In this work, liposomes (Lip) were used for non-viral, in vivo gene transfer of the human FIX gene into mouse organs. Plasmid DNA, containing the human FIX cDNA under the control of the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was encapsulated in 1-2-microns multilamellar Lip composed of egg phosphatidylcholine (EPC). The percentage of Lip-associated DNA was 47%, and 72% of the Lip DNA was protected from DNase I digestion. The Lip-encapsulated (Len) DNA was injected intravenously into Balb/c mice, and at various times post-injection, various tissues were examined for the presence of the exogenous DNA. Plasmid DNA was detected by Southern blot analysis mainly in the liver and spleen, but small amounts were also detected in the lungs, heart and kidneys. The plasmid DNA was retained in mouse liver cells for at least 7 days post-injection, and remained in an episomal state. The levels of human FIX protein in the mouse plasma were 190-650 pg per ml for 2 to 7 days post-injection. Treatment of mice with chloroquine (Cq) and colchicine (Cc) prior to Lip injection significantly increased the amount of plasmid DNA found in the liver cells, as well as the level of human FIX in the plasma. These results demonstrate the potential use of Len DNA for gene transfer into liver and spleen, and for gene therapy of inherited and acquired disorders.

Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle.

We have used retroviral vectors to introduce human or canine factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.

Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.

Toward the goal of gene therapy, we have been attempting to establish model somatic cell systems with the potential of sustained expression of the foreign gene. We report here that long-term expression of foreign genes in mouse embryo fibroblast implants can be achieved if a housekeeping gene promoter is used to drive transcription. Specifically, we have shown that in implants containing a beta-galactosidase gene linked to either an immediate early promoter of cytomegalovirus or a dihydrofolate reductase (DHFR) gene promoter, only the DHFR promoter allows long-term expression. We propose that choice of the promoter manifests significant influence on the long-term expression of genes introduced in fibroblast implants by retroviral vectors.

Phenotypic correction of factor IX deficiency in skin fibroblasts of hemophilic dogs.

Primary skin fibroblasts from hemophilic dogs were transduced by recombinant retrovirus (LNCdF9L) containing a canine factor IX cDNA. High levels of biologically active canine factor IX (1.0 micrograms per 10(6) cells per 24 hr) were secreted in the medium. The level of factor IX produced increased substantially if the cells were stimulated by basic fibroblast growth factor during infection. Additionally, we also report that endothelial cells transduced by this virus can produce high levels of biologically active factor IX. We propose that skin fibroblasts and endothelial cells from hemophilia B dogs may serve as potential venues for the development and testing of models for treatment of hemophilia B by retrovirally mediated gene replacement therapy.

Human and murine urokinase cDNAs linked to the murine alpha A-crystallin promoter exhibit lens and non-lens expression in transgenic mice.

cDNAs encoding either the human or the murine urokinase-type plasminogen activator (uPA) were fused downstream from the promoter-enhancer element of the murine gene encoding alpha A-crystallin, a protein found exclusively in the ocular lens. The DNAs were microinjected into fertilized mouse eggs as linear fragments free of bacterial sequences, and for each construct one line of transgenic mice was generated. In both lines transgenic uPA activity was detected in the ocular lens, in agreement with previous results reported on transgenic mice bearing genes fused to the same regulatory region. Unexpectedly however relatively high levels of this activity were found also in the retina, and furthermore, human uPA activity was found also in different parts of the brain and in the bone marrow, and to a lesser extent in the spleen, thymus and optic nerve. Transgenic uPA transcript was found in the lens, retina, brain and thymus of mice carrying the murine cDNA. Such a pattern of expression was different from that exhibited by the endogenous murine uPA gene and, excluding the lens, it appeared to be conferred by the cDNAs. The putative regulation by uPA cDNAs is suggested to be mediated through an internal enhancer-like element functioning in combination with the alpha A-crystallin promoter in a fashion independent of the specific nature of the promoter.

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Carcinosarcoma of the fallopian tube.

A patient with Stage III fallopian tube carcinosarcoma treated with cisplatin, doxorubicin, and cyclophosphamide, surviving 31 months disease-free after diagnosis, is presented. Review of the literature revealed this is the 28th case report of malignant mixed Mullerian tumor of the fallopian tube, and fifth instance in which chemotherapy was used as a primary therapy. Modern chemotherapy appears to have improved survival.

Therapy of ovarian carcinoma: the relationship of dose level and treatment intensity to survival.

Following primary maximal cytoreduction, 71 previously untreated patients with advanced epithelial ovarian carcinoma received at least six courses of combination chemotherapy consisting of cisplatin, doxorubicin, and cyclophosphamide. The cumulative dose (CD) through three (CD3) and six (CD6) courses was calculated for each drug and for all drugs combined. The dose intensity (DI) through three (DI3) and six (DI6) courses was calculated for each drug by dividing CD3 and CD6 by the interval (in weeks) between surgery and the third and sixth course. The interval from surgery to the third or sixth course had no effect on survival. Similarly, there was no significant difference in survival between patients with high and low CD3 or CD6 for any drug or for all drugs combined. Patients with high DI6 for cisplatin, doxorubicin, and all drugs combined survived significantly longer than those with low DI6. The survival difference for patients with high and low DI6 for cyclophosphamide approached, but did not attain, statistical significance at the 0.05 level. The intensity with which combination chemotherapy is administered may have an impact upon survival in patients with ovarian carcinoma.

Exfoliative dermatitis: presenting sign of fallopian tube carcinoma.

A woman presented with severe exfoliative dermatitis and a pelvic mass subsequently found to be fallopian tube carcinoma. After resection of the tumor and four courses of cisplatin, doxorubicin, and cyclophosphamide, the skin condition cleared.

Serum copper in ovarian carcinoma.

The serum copper and CA 125 levels of 31 patients with epithelial ovarian carcinoma were determined. Serum copper was elevated in seven patients and CA 125 was elevated in 22 patients. A rise in serum CA 125 always was associated with disease progression. In comparison, serum copper fluctuation did not correlate with the natural history of the malignancy. We concluded that serum copper determination has no use in epithelial ovarian carcinoma management.

Glassy cell carcinoma of the fallopian tube. A case report.

A 31-year-old woman, 10 weeks postpartum, presented with a right adnexal mass. The neoplasm was found to originate from the right fallopian tube and a right salpingoophorectomy was performed. Pathological examination found an adenosquamous carcinoma with features characteristic of a glassy cell carcinoma as described in the uterine cervix. The finding of this neoplasm in the fallopian tube suggests that it may be a tumor type common to the entire müllerian system.

Induction of urokinase-type plasminogen activator by UV light in human fetal fibroblasts is mediated through a UV-induced secreted protein.

Plasminogen activator was previously shown to be induced by UV light in human cells with low capacity to repair UV-induced DNA lesions. We now show that in human fetal fibroblasts UV light enhanced the two mRNA species coding for the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator, but immunological analysis revealed exclusively uPA activity. Several independent and complementary experiments indicated that induction of uPA was mediated, apparently entirely, through a UV-induced, secreted protein (UVIS) in the growth medium of irradiated cells. First, elevation of uPA mRNA after irradiation was severely blocked by cycloheximide. Second, replacement of conditioned medium in irradiated cells while the rate of plasminogen activator induction was maximal rapidly and completely stopped any further increase in uPA activity. Third, addition of the same removed conditioned medium to nonirradiated cells mimicked UV light in enhancing the level of uPA activity as well as that of uPA mRNA. Fourth, UVIS activity was completely lost by treating the conditioned medium with trypsin but not with nucleases. Kinetic measurements indicated that the accumulation of UVIS rather than the induction of uPA by UVIS conferred the rate-limiting step in the overall process of uPA induction. Both UV light and UVIS acted synergistically with inhibitors of DNA repair for uPA induction. Based on these results, a model is proposed implicating relaxation of DNA torsional stress of an as yet undefined DNA sequence(s) in the induction of UVIS, which is then responsible for activation of the uPA gene.

Multiple primaries among gynecologic malignancies.

Seventy-eight synchronous or metachronous tumors among 2362 patients followed by the Downstate Gynecologic Tumor Registry are reviewed. Significant synchronous tumor pairs include cervix (invasive and in situ)-ovary, cervix (in situ)-uterus, cervix (in situ)-kidney, endometrium-ovary, endometrium-rectosigmoid, and ovary-breast. Significant metachronous pairs include cervix (invasive and in situ combined)-lung, cervix (invasive and in situ combined)-upper alimentary tract, and cervix (invasive)-rectosigmoid. In the case of in situ and invasive cervical cancer-lower genital tract, significance was determined for both synchronous and metachronous pairs. Long survival is an important factor in the appearance of a second tumor as demonstrated in patients with cervical carcinoma. Synchronous data prove to be valuable in assessing in risk of second primaries in patients surviving for short periods. The roles of cigarette smoking, hormones, immunosuppression, radiotherapy, and screening are discussed.

Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1.

We isolated six temperature-sensitive mutants of poliovirus type 1 (Mahoney) by hydroxylamine mutagenesis and replica plating at 31, 33 (permissive), and 39 degrees C (restrictive). One of these mutants, designated tsB9, was chosen for more detailed examination. tsB9 accumulated 25% of the wild-type amount of virus-specific RNA at the restrictive temperature. We found that tsB9 was not able to synthesize mature, 35S single-stranded RNA at the restrictive temperature. In spite of the absence of significant RNA synthesis, tsB9 retained the ability to inhibit host protein synthesis during infection at 39 degrees C at about the same rate as wild-type virus.