Surgery for endometrial cancers with suspected cervical involvement: is radical hysterectomy needed (a GOTIC study)?

BACKGROUND: Radical hysterectomy is recommended for endometrial adenocarcinoma patients with suspected gross cervical involvement. However, the efficacy of operative procedure has not been confirmed. METHODS: The patients with endometrial adenocarcinoma who had suspected gross cervical involvement and underwent hysterectomy between 1995 and 2009 at seven institutions were retrospectively analysed (Gynecologic Oncology Trial and Investigation Consortium of North Kanto: GOTIC-005). Primary endpoint was overall survival, and secondary endpoints were progression-free survival and adverse effects. RESULTS: A total of 300 patients who underwent primary surgery were identified: 74 cases with radical hysterectomy (RH), 112 patients with modified radical hysterectomy (mRH), and 114 cases with simple hysterectomy (SH). Median age was 47 years, and median duration of follow-up was 47 months. There were no significant differences of age, performance status, body mass index, stage distribution, and adjuvant therapy among three groups. Multi-regression analysis revealed that age, grade, peritoneal cytology status, and lymph node involvement were identified as prognostic factors for OS; however, type of hysterectomy was not selected as independent prognostic factor for local recurrence-free survival, PFS, and OS. Additionally, patients treated with RH had longer operative time, higher rates of blood transfusion and severe urinary tract dysfunction. CONCLUSION: Type of hysterectomy was not identified as a prognostic factor in endometrial cancer patients with suspected gross cervical involvement. Perioperative and late adverse events were more frequent in patients treated with RH. The present study could not find any survival benefit from RH for endometrial cancer patients with suspected gross cervical involvement. Surgical treatment in these patients should be further evaluated in prospective clinical studies.

A novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer.

When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1-3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1-3 segment, and by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, alpha1-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1-3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo.

Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

Effects of glucocorticoid on body growth and serum levels of somatomedin A in the rat.

Serum levels of somatomedin A, as measured by radioreceptor assay, and body weight gain were 86.5 +/- 9.2% and 166.9 +/- 7.8% (N = 5) of the initial values, respectively, after 18 days administration of 2.5 mg cortisone acetate (CA). These values were significantly lower than those for saline treated rats (P less than 0.005). Reduced serum somatomedin A and body growth rate were partially restored after halting the injection of CA. Combined administration of daily doses of 100 micrograms hGH with CA did not prevent the decrease in somatomedin activity in treated rats. This observation suggests that GH plays a minor (or no) role in the fall of serum somatomedin A in CA-treated rats. From these data we conclude that glucocorticoids reduce serum somatomedin by inhibiting the effect of GH on the generation of somatomedin.

Effect of food and GH on the binding of 125I-somatomedin A to the membranes of rat lung and kidney.

Studies were undertaken to examine the relationship between serum levels of somatomedin A and its binding sites in the lung and kidney of rats. After three days of fasting, serum levels of somatomedin A decreased, accompanied by increases of 17.5% and 21.4% in binding to the plasma membranes from lung and kidney, respectively. After two days of refeeding, this binding decreased with the increase in the serum level of somatomedin A. There was observed a negative correlation between the serum level of somatomedin A and the percentage of labelled somatomedin A bound to the membranes. These observations suggest that somatomedin down-regulates its own receptor under these conditions. A similar regulatory mechanism was not observed with either hypophysectomized- or GH-treated rats.

Changes in serum somatomedin A and its binding to kidney membranes in growing rats.

Changes in serum somatomedin A levels and [125I]somatomedin A binding to membrane fractions from kidney were studied in rats 1-80 days of age. The mean level of serum somatomedin A was 0.80 U/ml at birth and increased with age; at 80 days the mean level was 7.41 +/- 0.67 U/ml. There was a close correlation between serum levels of somatomedin A and body weight. Labelled somatomedin A binding to membrane fractions from kidney was highest at birth and decreased with age up to 50 days. In Scatchard analysis of the data the affinity constant did not show a clear change with age, but the binding capacity decreased with age up to 30 days. An inverse correlation was observed between serum somatomedin A levels and labelled somatomedin A binding to membrane fractions from kidney. Compared to changes in circulating somatomedin A, the change in tissue binding was modest. The observation suggests that other circulating growth factors not measured by this radioreceptor assay or altered post-receptor sensitivity to somatomedins may be involved in growth.

Effect of insulin and nutrition on serum levels of somatomedin A in the rat.

The serum levels of somatomedin A, as measured by radioreceptor assay, were significantly reduced in rats 2 days after the administration of streptozotocin. The mean decrease was 45.4 +/- 2.9% of the initial values. In rats treated with insulin, blood glucose levels and glycosuria decreased, and serum somatomedin A returned to 108.3% +/- 11.7% of the initial values by the sixth day of treatment. In untreated diabetic rats, serum somatomedin A decreased progressively to 23.4 +/- 4.4% 8 days after streptozotocin administration. The total caloric intakes in the treated and nontreated diabetic rats were similar, suggesting that the low levels of somatomedin A in diabetic rats may be due to lack of insulin. A significant correlation was observed between serum somatomedin A values and body weight (r = 0.90) or the urinary glucose (r = -0.84) or blood glucose levels (r = -0.67). When the diabetic insulin-treated rats were fed a low protein diet, there was no increase in serum somatomedin A. Inhibitory factors in serum which interfere in the bioassay for somatomedin had no effect in our radioreceptor assay.

Effect of thyroid hormone on the serum level of somatomedin A.

The effect of thyroid hormone on the serum level of somatomedin A was studied using a radioreceptor assay for somatomedin A. In 13 patients with hyperthyroidism, the mean level of somatomedin A was 0.06 +/- 0.05 U/ml, which was within the normal range. Low levels were found in 19 patients with primary hypothyroidism with a mean of 0.39 +/- 0.04 U/ml. After treating these patients with thyroxine, the serum somatomedin A levels returned to the normal range. There was a significant correlation between the somatomedin A level on one hand and T3 (r = 0.49) and T4 (r = 0.55) level on the other. The serum level of somatomedin A did not decrease for 7 days after thyroidectomy nor increase after treating hypophysectomized rats with 2 microgram of thyroxine for 14 days. However, the effect of growth hormone on serum somatomedin A was augmented 42.2% by the combined administration of the same amount of T4 for 7 days. There was a positive correlation between the serum level of somatomedin A and the percentage increase in body weight (4 = 0.73). These data suggest that thyroxine potentiates the effect of GH on synthesis and/or the secretion of somatomedin A.

Effect of nutrition on growth and somatomedin A levels in the rat.

Serum somatomedin A was significantly reduced after 3 days of fasting in rats with a mean decrease of 23.6 +/- 2.4% (N = 18) of initial values. Re-feeding for one day produced a definite increase in somatomedin A, with a rise in body weight. When re-fed isocalorically for 21 days with diets of different quality, a low protein diet led to smaller increases in both serum somatomedin A and body weight in comparison to those of control-, high-protein- and high fat-diets (P < 0.001). There is a positive correlatin between the increase in body weight and serum somatomedin A levels (N = 70, r = 0.71, P < 0.001). The effect of growth hormone on somatomedin generation was abolished in hypophysectomized rats fed with low-protein diet. Our study suggests that protein in the diet is important for the generation of somatomedin A, which is necessary for normal growth.

Serum levels of somatomedin A and growth during long-term treatment of patients with pituitary dwarfism with human growth hormone.

Eighteen patients with pituitary dwarfism were treated for 1 7/12 to 6 years with human growth hormone (hGH) at a dose of 0.19--0.62 unit (U) per kg of body weight per week. The mean increment in height was 2.0 +/- 0.4 and 8.8 +/- 0.5 cm/year before and the first year after treatment of hGH, respectively. A significant positive correlation was observed between serum levels of somatomedin A and growth rate, espcially in children with bone age below 10 and a duration of treatment of less than one year (r = 0.66, P less than 0.005). Long-term treatment with hGH was accompanied by a decreasing response. However, the serum levels of somatomedin A did not decrease significantly. Therefore, decreased growth increment in these situations was not due to decreased serum levels of somatomedin A.