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ABSTRACT: The nuclear phosphoprotein p53 is expressed in all normal cells and appears to function in cell cycle regulation. Abnormally high levels of the protein are found in many different types of cancer. In breast carcinoma overexpression of p53 is associated with point mutations within highly conserved regions of the p53 gene. These altered genes encode stable p53 proteins that can be detected by standard immunohistochemical techniques unable to detect rapidly degraded wild-type protein. The level of p53 expression in 184 primary breast cancer specimens was assessed by immunohistochemical analysis and related to the following established prognostic factors for breast cancer: age, stage, metastatic involvement, concentration of estrogen and progesterone receptors, proliferative index, and HER-2/neu overexpression. Fifty (27%) of these primary breast cancer specimens had widespread overexpression of p53. Highly significant associations were found between p53 overexpression and late stage, metastatic spread, and low concentration of progesterone receptors. The presence of elevated levels of mutant p53 may itself be a prognostic factor in human breast cancer and activation of this oncogene may be important in the ability of a tumor to metastasize.
Surgery 09/1991; 110(2):259-64. · 3.10 Impact Factor
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ABSTRACT: We examined p53 expression in 107 epithelial ovarian cancers with immunohistochemical techniques using monoclonal antibody PAb1801. High level expression of nuclear p53 protein was detected in the malignant epithelium in 54 (50%) of these cancers. Expression of p53 protein was undetectable in 13 benign gynecological tissues. p53 mRNA from three cancers that overexpressed the protein were sequenced and point mutations which altered the coding sequence of the highly conserved region of the gene were found in each case. Three cancers with undetectable protein levels also were sequenced and were found to be wild-type through the same region of the gene. As in other cancers, overexpression of the p53 protein in ovarian cancer appears to correlate closely with the presence of mutation in the p53 gene. p53 overexpression did not correlate with stage, histological grade, or the ability to perform optimal cytoreductive surgery. A significant correlation (P = 0.04) was observed between p53 overexpression and aneuploidy in advanced stage (III/IV) disease. There was no significant relationship between overall survival and p53 expression. Since mutation and overexpression of p53 are common in epithelial ovarian cancers, further studies are warranted to clarify the role of p53 in ovarian tumorigenesis.
Cancer Research 07/1991; 51(11):2979-84. · 7.86 Impact Factor
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ABSTRACT: Overexpression of an activated form of the p53 protein may be involved in neoplastic transformation. We found widespread overexpression of p53 by immunohistochemical staining in 11 (22%) of 49 primary invasive human breast cancers. Northern blot analysis showed that this overexpression was not due to an increase in the steady-state level of p53 mRNA. The p53 gene was directly sequenced in 7 of these tumors with elevated levels of the protein and, in each case, a mutation that altered the coding sequence for p53 was found in a highly conserved region of the gene. Whereas 4 of these tumors contained only a mutant p53 allele, the other 3 tumors exhibited coding sequences from both a mutant and a wild-type allele. p53 mutations have previously been correlated with allelic loss of part of chromosome 17p that contains the p53 locus. Examination of all 49 breast tumors revealed a 61% frequency of deletion at or near the p53 locus. However, the presence of allelic deletion did not correlate with overexpression of the protein. Six tumors that were deleted but did not express high levels of the protein were sequenced and all retained a wild-type p53 allele. In this series of human breast cancers, overexpression of the p53 protein, not allelic loss on chromosome 17p, was always associated with mutation of the p53 gene.
Proceedings of the National Academy of Sciences 07/1991; 88(11):5006-10. · 9.68 Impact Factor
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ABSTRACT: Overexpression of the nuclear phosphoprotein p53 is one of the most common abnormalities in primary human cancer and appears to be due to point mutation within a highly conserved region of the p53 gene which then encodes for a mutant, more stable protein. In this study different stages of breast cancer progression were examined, from in situ to metastatic disease, to determine at what stage mutational activation occurs and whether it is maintained during tumor progression. Two (13%) of 15 pure intraductal tumors expressed high levels of p53 in all malignant epithelial cells. Sequencing of p53 mRNA from one of these tumors demonstrated a nucleotide substitution altering the amino acid composition of the protein. Six (17%) of 35 specimens which contained both in situ and invasive disease expressed high levels of p53. All malignant epithelial cells in these 6 cases stained positively and in no specimen did one component express different levels of the protein than the other growth phase. Sequence analysis of a tissue with significant amounts of both in situ and invasive disease revealed only a single point mutation, without evidence of wild-type nucleotide at the site of substitution, suggesting that p53 mRNA from each component of the tumor contained the same nucleotide substitution. Eleven (50%) of 22 pairs of primary tumors and their lymph node metastases expressed elevated levels of p53, and in each case, expression levels were identical in the primary and secondary sites. Identical mutations were found in the p53 mRNA from two paired primary and metastatic sites. Therefore, mutation within a highly conserved region of the p53 gene leading to overexpression of the protein product can occur in the earliest recognized phase of breast cancer and this alteration is maintained during progression from intraductal to infiltrating carcinoma. Mutations are also conserved during the process of metastatic spread.
Cancer Research 06/1991; 51(10):2605-10. · 7.86 Impact Factor
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ABSTRACT: Pneumatosis intestinalis (PI) occurs in a wide variety of patients, some of whom require urgent surgery, while others can be observed with resolution of symptoms and radiographic findings. During 1 year, 27 patients with PI were prospectively evaluated for clinical, laboratory, and radiographic features that would be useful in predicting the need for surgery, the pathologic findings, and patient outcome. Sixteen of the twenty-seven patients underwent laparotomy, with only one negative exploration. Of the 11 patients not explored, there were two deaths in moribund patients. Seven of nine patients with jejunostomy tubes, recent gastrointestinal anastomoses, inflammatory bowel disease, lactulose therapy, or chemotherapy who did not have clinical evidence of an acute surgical abdomen or metabolic acidosis survived without surgery (two deaths unrelated to the gastrointestinal tract). Patients presenting with bowel obstruction and PI required surgery in seven of nine cases, did not have necrotic bowel, and had 11% mortality. Eight patients with ischemic bowel had a 75% mortality rate, despite surgery. Patients with PI and clinical evidence of bowel obstruction or ischemia usually require urgent surgery, while asymptomatic patients without metabolic acidosis can be safely observed.
Annals of Surgery 09/1990; 212(2):160-5. · 7.49 Impact Factor
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ABSTRACT: The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1 x 1012 vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1 ± 0.5 µg/mL, or 22% of normal) that were comparable to those achieved after direct delivery of the same vector dose into the portal circulation (1.3 ± 0.3 µg/mL, or 26% of normal). Importantly, the pattern of vector biodistribution after systemic and portal vein administration of scAAV-LP1-hFIXco was almost identical. Additionally, comparable levels of gene transfer were achieved in macaques with preexisting immunity to AAV8 following peripheral vein administration of 1 x 1012 vg/kg AAV5-pseudotyped scAAV-LP1-hFIXco. This confirms that alternative serotypes can circumvent preexisting naturally acquired immunity to AAV. Thus, peripheral venous administration of AAV5 and AAV8 vectors is safe and as effective at transducing the liver in nonhuman primates as direct vector administration into the portal circulation. These results should make vector administration to patients, especially those with a severe bleeding diathesis, significantly easier and safer.
Nathwani, A.C. and Gray, J.T. and McIntosh, J. and Ng, C.Y.C. and Zhou, J. and Spence, Y. and Cochrane, M. and Gray, E. and Tuddenham, E.G.D. and Davidoff, A.M. (2007) Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates. Blood, 109 (4). pp. 1414-1421. ISSN 00064971.
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ABSTRACT: Although significant progress has been made in the management of children with Wilms tumor, two major controversies still exist: the extent of radiographic evaluation necessary before surgery and the role of preoperative chemotherapy. This study sought to determine whether patients with Wilms tumor who presented with abdominal pain defined a special subset of patients who might require a more extensive preoperative work-up and neoadjuvant chemotherapy.
From 1970 to 1995, 250 children were treated for Wilms tumor at a single pediatric institution. A retrospective chart review determined presenting signs and symptoms for each patient.
Thirty-four (14%) patients (mean age 5.5 years) sought medical attention with a chief complaint of abdominal pain. The stage distribution for these patients tended to be higher and was significantly different (P <.001, chi(2)-analysis) from those presenting without pain. Four (14%) of 29 patients with tumor available for analysis were found to have anaplastic histology.
These data suggest that patients with Wilms tumor who present with abdominal pain represent a special subgroup that tends to be older and has an increased incidence of tumor rupture, anaplasia, and higher stage. These patients may benefit from a more extensive preoperative evaluation and consideration of neoadjuvant chemotherapy.
Annals of Surgical Oncology 5(3):213-5. · 4.17 Impact Factor
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A C Nathwani, A M Davidoff,
H Hanawa,
Y Hu,
F.A. Hoffer,
A. Nikanorov,
C Slaughter,
C.Y.C. Ng,
Z Zhou,
J.N. Lozier,
T.D. Mandrell,
E F Vanin,
A W Nienhuis
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ABSTRACT: The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.
Nathwani, A.C. and Davidoff, A.M. and Hanawa, H. and Hu, Y. and Hoffer, F.A. and Nikanorov, A. and Slaughter, C. and Ng, C.Y.C. and Zhou, Z. and Lozier, J.N. and Mandrell, T.D. and Vanin, E.F. and Nienhuis, A.W. (2002) Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques. Blood, 100 (5). pp. 1662-1669. ISSN 00064971.
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A M Davidoff,
C.Y.C. Ng,
Y Zhang,
C J Streck,
S.J. Mabry,
S.H. Barton,
T. Baudino,
J Zhou,
R.S. Kerbel,
E F Vanin,
A C Nathwani
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ABSTRACT: To inhibit tumor-induced angiogenesis, the VEGF signaling pathway was targeted using AAV vectors encoding a VEGF decoy receptor, a truncated, soluble form of the murine VEGF receptor-2 (tsFlk-1). This approach initially had significant anti-neuroblastoma efficacy in murine xenograft models of local and metastatic disease, but when higher circulating levels of tsFlk-1 were established, tumor growth was more aggressive than even in control mice. Part of the mechanism for this apparent tumor resistance was increased human VEGF expression by the tumor cells. However, further investigation revealed that although a greater amount of VEGF could be bound by higher levels of tsFlk-1, more VEGF also existed in an unbound state and was, therefore, available to support angiogenesis. This novel, tumor-independent mechanism for resistance to antiangiogenic strategies suggests that careful titering of angiogenesis inhibitors may be required to achieve maximal antitumor efficacy and avoid therapy resistance mediated, in part, by ligand bioavailability. This has important implications for therapeutic strategies that use decoy receptors and other agents, such as antibodies, to bind angiogenic factors, in an attempt to inhibit tumor neovascularization.
Davidoff, A.M. and Ng, C.Y.C. and Zhang, Y. and Streck, C.J. and Mabry, S.J. and Barton, S.H. and Baudino, T. and Zhou, J. and Kerbel, R.S. and Vanin, E.F. and Nathwani, A.C. (2005) Careful decoy receptor titering is required to inhibit tumor angiogenesis while avoiding adversely altering VEGF bioavailability. Molecular Therapy, 11 (2). pp. 300-310. ISSN 15250016.
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ABSTRACT: A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2– and AAV-5–based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5 dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.
Davidoff, A.M. and Ng, C.Y.C. and Zhou, J. and Spence, Y. and Nathwani, A.C. (2003) Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway. Blood, 102 (2). pp. 480-488. ISSN 00064971.
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