Darbepoetin alfa once every 2 weeks for treatment of anemia in dialysis patients: a combined analysis of eight multicenter trials.

AIM: Darbepoetin alfa has a longer half-life than epoetin-(EPO) alfa or beta, allowing administration at less frequent intervals for the treatment of renal anemia. The aim of the present analysis was to evaluate the efficacy and tolerability of an every-2-week (Q2W) schedule of darbepoetin alfa in a large cohort of dialysis patients. METHODS: Data were combined from eight similarly designed 24-week phase 3b European studies, in which patients receiving EPO alfa or beta once-weekly were converted to Q2W darbepoetin alfa. Darbepoetin alfa dosage was titrated to maintain hemoglobin (Hb) between 10 and 13 g/dl and efficacy was evaluated during a 4-week evaluation period. RESULTS: In the 1,101 patients assigned to Q2W darbepoetin alfa (i.v., n = 196, s.c., n = 905), mean (SD) Hb levels were 11.53 (0.77) g/dl at baseline and 11.35 (1.04) g/dl at evaluation (mean change in Hb -0.27 g/dl, 95% confidence interval 0.34, -0.20). Hb levels were maintained between 10 and 13 g/dl during evaluation in 85% of patients. Darbepoetin alfa doses were similar at baseline and evaluation, and the i.v. and s.c. routes were associated with similar efficacy and dose requirements. Darbepoetin alfa was well-tolerated. CONCLUSIONS: Q2W darbepoetin alfa is effective in maintaining Hb levels in dialysis patients switched from weekly rHuEPO, regardless of the route of administration and with no notable increase in the weekly equivalent dose.

Smallest region of overlapping deletion in 1p36 in human neuroblastoma: a 1 Mbp cosmid and PAC contig.

In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244. In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms. Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected. Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p. One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal). To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the I Mbp SRO consisting predominantly of bacteriophage P1-derived artificial chromosome (PAC) clones. A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp. Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO. On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis. Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO. The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma.

Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP.

The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm. In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses. Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP. Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library). Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively. The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype. The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain. A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma. However, further analyses are required to confirm these results.

Fine mapping of distal 1p loci reveals TP73 at D1S468.

In the present study we establish a FISH fine-map of 1p36.3 loci. This region is frequently altered in different types of human tumors suggesting the existence of cancer-related genes. Identification of cosmids carrying both D1S468 and TP73 sequences leads to the assignment of TP73 to the most frequently deleted locus in colon and breast cancer and integrates this gene in human genetic maps. Localization of other distal loci was determined as follows: distal-CDC2L1-D1Z2-D1S94-TP73/D1S468-D1 S1615-proximal. D1S1615, earlier reported as a telomeric sequence, is considerably more proximal than previously thought.

Cloning of the human aflatoxin B1-aldehyde reductase gene at 1p35-1p36.1 in a region frequently altered in human tumor cells.

Alterations of the distal portion of the short arm of chromosome 1 (1p) are among the earliest abnormalities of human colorectal tumors. Loss of heterozygosity analysis has previously revealed a smallest region of overlapping deletion (SRO) B, at 1p35-36.1, deleted in 48% of sporadic tumors. From this region we have now cloned a gene encoding a protein of 330 amino acids that is 78% identical with the Rattus norvegicus aflatoxin B1 aldehyde reductase (Afar) and, therefore, likely represents its human homologue. In rat liver, Afar is strongly inducible by the antioxidants ethoxyquin and butylated hydroxyanisole, which protect the rat against aflatoxin B1-induced liver tumorigenesis by detoxifying its genotoxic and cytotoxic dialdehyde. Human AFAR is expressed in a broad range of tissues and, therefore, is likely involved in endogenous detoxication pathways. Impaired detoxication of genotoxic aldehydes and ketones, which are involved in tumorigenesis of the colon and breast, may be a crucial factor both for tumor initiation and progression. We here provide a detailed contig of 1.5-2 Mbp/2.7 cM encompassing part of SRO B, including known genes and previously unmapped expressed sequence tags. PLA2G2A (secretory type II phospholipase A2), described previously as a candidate, is localized outside SRO B.

Secretory type II phospholipase A2 (PLA2G2A) expression status in colorectal carcinoma derived cell lines and in normal colonic mucosa.

There is good evidence now that the secretory type II phospholipase A2 (Pla2g2a) gene represents the Mom1 locus, a genetic modifier of tumor resistance in the multiple intestinal neoplasia (Min) mouse. Previously we have mapped the human homolog PLA2G2A to 1p35-36.1 within a region that is the target of frequent deletions in sporadic colorectal tumors. Here we show 64% loss of heterozygosity (LOH) at the PLA2G2A locus in primary tumors. We studied PLA2G2A expression in both colorectal tumor cell lines and normal mucosa. Most of the lines lacked detectable PLA2G2A transcripts by Northern analysis. Large differences in expression were seen among normal mucosa of different patients with sporadic tumors. We analysed the coding region of PLA2G2A in eight colorectal cancer cell lines with hemizygous deletion at 1p35-36/PLA2G2A, in none we did detect a mutation. Biallelic expression of PLA2G2A was observed in a cell line heterozygous for an exon 3 polymorphism, rendering unlikely that imprinting is a pathway participating in the loss of PLA2G2A function. It remains uncertain if PLA2G2A, in particular its apparent lack of expression in tumor cells, might be a factor in human colorectal tumorigenesis.

Genomic instability in 1p and human malignancies.

Both cytogenetic and molecular genetic approaches have unveiled non-random genomic alterations in 1p associated with a number of human malignancies. These have been interpreted to suggest the existence of cancer-related genes in 1p. Earlier studies had employed chromosome analysis or used molecular probes mapped by in situ hybridization. Further, studies of the various tumor types often involved different molecular probes that had been mapped by different technical approaches, like linkage analysis, radioactive or fluorescence in situ hybridization, or by employing a panel of mouse x human radiation reduced somatic cell hybrids. The lack of maps fully integrating all loci has complicated the generation of a comparative and coherent picture of 1p damage in human malignancies even among different studies on the same tumor type. Only recently has the availability of genetically mapped, highly polymorphic loci at (CA)n repeats with sufficient linear density made it possible to scan genomic regions in different types of tumors readily by polymerase chain reaction (PCR) with a standard set of molecular probes. This paper aims at presenting an up-to-date picture of the association of 1p alterations with different human cancers and compiles the corresponding literature. From this it will emerge that the pattern of alterations in individual tumor types can be complex and that a stringent molecular and functional definition of the role that Ip alterations might have in tumorigenesis will require a more detailed analysis of the genomic regions involved.

Human homologue of a candidate for the Mom1 locus, the secretory type II phospholipase A2 (PLA2S-II), maps to 1p35-36.1/D1S199.

Mice heterozygous for the dominant Min mutation in their Apc gene develop multiple intestinal neoplasia. Analogously, family members from familial adenomatous polyposis kindreds inheriting mutations in their human APC homologue develop a similar phenotype. Quantitative trait loci studies have identified the Mom1 locus (for modifier of Min-1), which is responsible for part of the genetic variability in polyp number found among inbred mouse strains. The secretory type II phospholipase [nonpancreatic Pla2s (type II Pla2s or Pla2s-II)] has been demonstrated to be a candidate for Mom1, and a mutation in Pla2s-II in mice carrying the Min mutation has been proposed to account for an increased polyp number compared to mice without the Pla2s-II mutation. In this study, we have mapped the chromosomal position of the human homologue of Pla2s-II. We have identified 3 mega-yeast artificial chromosomes that carry PLA2S-II and localized one of them by fluorescence in situ hybridization to the border between 1p35 and 1p36.1. The presence of the microsatellite marker D1S199 in all three clones integrates PLA2S-II into different genetic maps. This highly polymorphic CA repeat D1S199 has previously been shown by us to identify loss of heterozygosity in 48% of sporadic colorectal tumors, indicating that the human homologue of the Pla2s-II/Mom1 locus might be related to human colorectal cancer.

Deletion mapping defines different regions in 1p34.2-pter that may harbor genetic information related to human colorectal cancer.

Cytogenetic and molecular analyses of colorectal cancer cells have revealed deletions at 1p as prominent alterations, suggesting that genetic information on the short arm of chromosome 1 has a role in tumorigenesis. In this study we have used 33 microsatellite markers to fine map deletions at 1p in primary colorectal carcinomas. We found 1p-deletions in 84% of the cases (31/37). High frequencies of loss of heterozygosity (LOH), often the result of small independent interstitial deletions in the same tumor, defined three regions, that may harbor genetic information relevant for colorectal cancer: (i) region A between D1S243 and D1S468 (7cM; 1p36.3); (ii) region B between D1S436 and D1S199 (7cM; 1p35.1-36.31) and (iii) region C between D1S496 and D1S255 (1cM; 1p34.2-35). In addition we identified seven cell lines with LOH at 1p, all of which have deletions that span at least from the distal border of region A to the proximal border of region C.

A reciprocal translocation (1;15) (36.2;q24) in a neuroblastoma cell line is accompanied by DNA duplication and may signal the site of a putative tumor suppressor-gene.

Cytogenetic analyses and molecular deletion studies of human neuroblastomas have indicated the chromosomal bands 1p36.1-1p36.2 as a location of genetic information which may be involved in tumorigenesis. To define this putative neuroblastoma locus in more detail we have analysed cell lines with alterations of distal 1p. Here we show, by fluorescence in situ hybridization (FISH), that cell line NGP has a reciprocal 1;15 translocation. Loci D1S214/D1S96 could be shown to map telomeric/distal, D1S228 centromeric/proximal to the break. We have identified yeast artificial chromosomes (YACs) that cover the break and map to D1S160 and D1S244. This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumors (Weith et al., 1989; Caron et al., 1993; Schleiermacher et al., 1994) and within the region of a constitutional interstitial deletion of a neuroblastoma patient (Biegel et al., 1993). Mapping studies with FISH revealed that the translocation is associated with duplication of DNA. It appears, as if the subchromosomal region we describe here is a good candidate for harboring the postulated neuroblastoma suppressor-gene.

Reciprocal translocation at 1p36.2/D1S160 in a neuroblastoma cell line: isolation of a YAC clone at the break.

Band 1p36.1-1p36.2 is frequently involved in chromosomal aberrations of neuroblastoma cells, and therefore thought to harbour genetic information which may be involved in tumorigenesis. To map this putative neuroblastoma locus, we screened neuroblastoma cell lines for reciprocal translocations at 1p36.1-2 which may signal the site of an affected gene. We identified a reciprocal 1;15 translocation in cell line NGP by fluorescence in situ hybridisation (FISH). As a strategy to clone the translocation breakpoint, we isolated yeast artificial chromosomes (YACs) specific for loci at 1p36. Screening of cell line NGP by FISH identified a YAC, 1050 kbp in size, which hybridised to both derivative 1;15 and 15;1 chromosomes. We conclude that this YAC, which maps to D1S160, covers the break. This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumours and within the region of a constitutional interstitial deletion of a neuroblastoma patient. The YAC we describe here should serve as a DNA source for gene cloning approaches towards the isolation of candidates for the putative neuroblastoma suppressor gene.