Radiotherapy omitted in the treatment of selected children under 3 years of age with stage III favorable histology Wilms tumor.

BACKGROUND: Current treatment of stage III favorable histology (FH) Wilms tumor is surgery, radiotherapy to residual disease, and "triple" chemotherapy (vincristine, dactinomycin, and doxorubicin) for 12 months. This study tests the hypothesis that some stage III patients, especially very young children with minimal residual abdominal disease, might be successfully treated without radiotherapy, thereby avoiding the adverse late effects associated with radiotherapy. PROCEDURE: From 1984, radiotherapy was omitted from the treatment of 8 carefully selected children who were younger than 3 years of age at diagnosis with stage III Wilms tumor by virtue of microscopic residual disease after surgery and whose lymph nodes were not involved by tumors. They were followed with bimonthly abdominal ultrasound examinations to assess local control. RESULTS: Follow-up is now from 2 to 12 years (median 6 years) and 7 of the 8 children are alive and well with no abdominal recurrence. One child relapsed in the lungs and despite further treatment died of progressive disease. The disease-free survival (DFS) and overall survival (OS) are therefore both 87.5%. CONCLUSIONS: The DFS and OS in this admittedly small sample are consistent with the survival rates for stage III FH Wilms tumor in the first United Kingdom Children's Cancer Study Group (UKCCSG), North American (NWTS), and European (SIOP) Wilms Tumor studies, Larger numbers of patients are needed to determine whether or not this treatment approach is generally applicable, but we conclude that some children in this stage III "substage" may be treated successfully without radiotherapy.

Retinal and choroidal neovascularization in a transgenic mouse model of sickle cell disease.

A complication of sickle cell disease is proliferative retinopathy. We investigated the eyes from a transgenic mouse model of sickle cell disease (alpha H beta S[beta MDD] type) to determine if pathological changes occurred in their retinas and choroids. One retina from each animal was processed by flat-embedding adenosine diphosphatase-reacted retinas in glycol methacrylate. The fellow eye from each animal was embedded whole in glycol methacrylate for histopathological analysis of all ocular structures. Retinal vascular occlusions resulted in nonperfused areas of retina and arterio-venous anastomoses. Intra- and extraretinal neovascularization was observed adjacent to nonperfused areas. Retinal pigmented lesions were formed by the migration of retinal pigment epithelial cells into sensory retina, often ensheathing choroidal neovascularization. The incidence of this bilateral chorioretinopathy was 30% in animals older than 15 months of age. The ocular histopathological changes we observed in the mouse model mimicked many aspects of human proliferative sickle cell retinopathy. Furthermore, this is the first genetically derived animal model for chorio-retinal neovascularization.

Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria.

The polymorphic frequency of the gene for beta s-globin involved in the generation of sickle trait and sickle cell anemia in the human population is caused by the enhanced resistance of sickle trait individuals to Plasmodium falciparum malaria, as supported by epidemiologic and in vitro studies. However, the mechanism for the protective effect of sickle hemoglobin in vivo has not been fully defined. The generation of transgenic mice expressing high levels of human beta s- and alpha-chains has allowed us to study this phenomenon in vivo in an experimental model. We infected the transgenic beta s mice with two species of rodent malaria and found a diminished and delayed increase in parasitemia as compared with controls. This is in contrast to our previous studies involving the introduction of a beta A transgene, which does not alter the infection. The use of this model allowed us to address the question of the mechanism of protection against malaria in mice expressing sickle hemoglobin. We find that splenectomy of transgenic mice completely reverses the protection against Plasmodium chabaudi adami infection. The results reported have shown a relationship between the presence of the beta s gene product and partial resistance to malaria in an experimental model in vivo and shows that the spleen plays an important role in this protection.

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.

High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia.

A line of transgenic mice with two cointegrated transgenes, the human beta S- and alpha 2-globin genes, linked to the beta-globin locus control region was produced and bred with mice carrying a deletion of the mouse beta major-globin gene. In transgenic mice homozygous for the beta major deletion (alpha H beta S[beta MDD]; where alpha H is human alpha-globin and MD is mouse deletion), 72.5 +/- 2.4% (mean +/- SD) of the beta-chains are beta S and the ratio of alpha H- to beta S-globin was 0.73. Introduction of a heterozygous mouse alpha-globin deletion into mice homozygous for the beta major deletion (alpha H beta S[alpha MD beta MDD]) resulted in 65.1 +/- 8.5% beta S and a human alpha/beta ratio of 0.89 +/- 0.2. Sickling occurs in 95% of erythrocytes from alpha H beta S[beta MDD] mice after slow deoxygenation. Transmission electron microscopy revealed polymer fiber formation but not fascicles of fiber. Increased organ weight was noted in lung, spleen, and kidney of transgenic mice vs. controls that may be due to hypertrophy or increased blood volume in the lungs and/or increased tissue water content. The hemoglobin content of lung, spleen, and kidney was also elevated in transgenic animals due to trapped hemoglobin and/or increased blood volume. When transgenic and control mice were examined by magnetic resonance imaging at 9.4 tesla, some transgenic animals had enlarged kidneys with prolonged relaxation time, consistent with increased organ weight and water content. The glomerular filtration rate was elevated in transgenic animals, which is characteristic of young sickle cell patients. Furthermore, exposure to hypoxia resulted in significantly decreased hematocrit, increased erythrocyte density, and induced a urine-concentrating defect. We conclude that the transgenic mouse line reported here has chronic organ damage and further hematological and organ dysfunction can be induced by hypoxia.

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.

The human homolog of the Moloney leukemia virus integration 2 locus (MLV12) maps to band p14 of chromosome 5.

The Moloney leukemia virus integration 2 (MLV12) locus represents a common region for proviral integration and a putative oncogene involved in the induction of thymic lymphomas in rodents. The human homolog of the MLV12 locus has been cloned and studies have been initiated to determine its possible role in the induction and progression of human neoplasms. In this study we used a panel of human X rodent somatic cell hybrids and in situ hybridization to metaphase chromosomes to map MLV12 to the short arm of the human chromosome 5, band p14.

Insertion of an Alu SINE in the human homologue of the Mlvi-2 locus.

Fifty-nine human DNA samples derived from either normal tissues or hematopoietic neoplasias were examined for rearrangements in the Mlvi-2 locus, a putative oncogene. The rearranged Mlvi-2 sequences in one of them, a B cell lymphoma, were shown to result from the insertion of an approximately 300 bp DNA fragment that hybridized to a human Alu probe. DNA sequence analysis of both the rearranged and the nonrearranged allele around the site of the insertion revealed the following: a) the insert was 88.4% homologous to the consensus sequence of the Alu family of repeats and 75% homologous to the Alu related sequence in the human 7SL RNA; b) similar to other sequenced SINES, a poly(d.A) tract was present at the 3' end of this element; c) an 8 bp direct repeat was present at both ends of the inserted element; d) this repeat was present as a single copy in the unrearranged allele. We conclude from these findings that: Alu sequences can transpose and that the direct repeats flanking certain Alu SINES may be generated by the duplication of single copy cellular sequences at the site of the insertion. Furthermore the recent nature of the Alu insertion in the Mlvi-2 locus coupled to the low degree of homology of the inserted Alu to the Alu related sequence in the 7SL RNA suggest that this event did not occur via reverse transcription and reintegration of the 7SL RNA.

Insertion of long interspersed repeated elements at the Igh (immunoglobulin heavy chain) and Mlvi-2 (Moloney leukemia virus integration 2) loci of rats.

Restriction enzyme analysis of normal DNA derived from individual rats of the National Institutes of Health outbred Osborn-Mendel colony revealed that two independent single-copy loci, the Igh (immunoglobulin heavy chain) locus and the Mlvi-2 (Moloney leukemia virus integration 2) locus, a putative oncogene, are polymorphic (i.e., exhibit allelic variation). The polymorphism at both loci was due to the presence or absence of a long interspersed repeated DNA element (LINE). The LINE insertion in the Igh locus occurred in the joining (J) region, which is involved in the physiological rearrangement of this locus. The LINE insertion in the Mlvi-2 locus has occurred approximately 6 kilobases from the region of provirus integration in Moloney murine leukemia virus-induced rat thymomas. The two inserts are colinear with each other and with other randomly selected cloned copies of the rat LINE family, the general characteristics of which we also present. LINE insertion in the Mlvi-2 locus was observed in several rat strains, established from independent rat colonies, suggesting that LINE-containing Mlvi-2 alleles may be widespread in the rat population. LINE insertion in the Igh locus was observed in 1 of 27 rats. The detection of a LINE-related polymorphism at two nonselected loci indicates that LINEs are transposable. The presence or absence of these long (greater than 5 kilobases), highly transcribed elements at single-copy loci could have profound effects on gene activity. Furthermore, LINE-containing single-copy loci could be affected by homologous interaction between the resident LINE and any of the other 50,000 or so copies of these elements in the rat genome.