Epidermal growth factor receptor extracellular domain mutations in primary glioblastoma.

AIMS: Novel missense mutations of the epidermal growth factor receptor (EGFR) extracellular domain have been recently described in a large series of glioblastomas. METHODS: The exons 2, 3, 7, 8 and 15 coding for the EGFR extracellular domain were sequenced in a series of 161 consecutive primary glioblastomas and correlated with clinical features of patients in order to determine whether these alterations are linked to specific clinical characteristics of the disease. RESULTS: Missense mutations were observed in 18 cases (11.2%), and 4 novel mutations were detected, including G178C, A271C, C818A and C1860G. Mutations of the EGFR extracellular domain were not associated with overall survival or with age at onset of the disease. In contrast, the EGFR extracellular domain mutations were significantly associated with patients' gender. Indeed, 15 mutations were observed in men vs. 3 in women (P < 0.05). EGFR extracellular domain mutations were also strongly associated with EGFR amplification (P < 0.0001). CONCLUSIONS: To our knowledge, EGFR extracellular domain mutations are the first genomic abnormalities associated with gender in primary glioblastomas, although a link between mutations of the EGFR tyrosine kinase domain and gender has been previously made in lung cancer.

TP53 codon 72 polymorphism is associated with age at onset of glioblastoma.

BACKGROUND: Functional single nucleotide polymorphisms (SNP) in codon 72 of TP53 have been shown to be a risk factor, a prognostic marker, and related factor to age at onset in various cancers. METHODS: We investigated blood samples from 254 patients with glioblastoma and 238 healthy controls. RESULTS: TP53 codon 72 status was not correlated with prognosis and did not differ between glioblastoma and control populations. However, the Pro/Pro genotype was overrepresented in patients <45 years (20.6% vs 6.4% in patients with glioblastoma >45 years, p = 0.002, vs 5.9% in control group, p = 0.001). We then confirmed this result on an independent series of young patients with glioblastoma. Finally, the analysis of tumor DNA found the Pro allele associated with occurrence of TP53 somatic mutation. CONCLUSION: Our data suggest that TP53 functional variation is particularly critical for oncogenesis of glioblastoma in young patients.

alpha-Internexin expression identifies 1p19q codeleted gliomas.

BACKGROUND: alpha-Internexin (INA) is a proneural gene encoding a neurofilament interacting protein that is upregulated in some gliomas, particularly oligodendrogliomas. METHODS: INA expression was evaluated by immunohistochemistry in a series of 122 gliomas, and correlated to the 1p19q codeletion, a favorable prognostic marker of oligodendroglial tumors. RESULTS: INA expression was strong (>10% positive cells) in 22 cases (22 oligodendroglial tumors and 0 astrocytic tumors), weak (<10% cells) in 14 cases (12 oligodendroglial tumors, 2 glioblastoma with an oligodendroglial component, and 0 astrocytic tumors), and negative in 86 cases (49 oligodendroglial tumors, 9 glioblastoma with an oligodendroglial component, and 28 astrocytic tumors). Among the 27 tumors exhibiting the 1p19q codeletion (all with an oligodendroglial phenotype), INA was detected in 96% (26/27, 18 strong, 8 weak) as compared to 11% (10/95, 4 strong, 6 weak) in the tumors without 1p19q codeletion (with an oligodendroglial or an astrocytic phenotype) (p < 0.001). In oligodendroglial tumors, INA expression specificity for 1p19q codeletion was 86%, sensitivity 96%, positive predictive value 76%, and negative predictive value was 98%. The prognostic impact of INA expression could be evaluated in grade III oligodendroglial tumors. Similar to 1p19q deletion, positive INA expression was correlated with better progression-free survival (52.6 vs 8.7 months [p = 0.001]) and overall survival (121.1 vs 31.4 months [p = 0.0001]). CONCLUSION: alpha-Internexin (INA) expression appears to be a simple, reliable prognostic marker and a surrogate marker of 1p19q codeletion.

Genetic markers predictive of chemosensitivity and outcome in gliomatosis cerebri.

BACKGROUND: Up-front temozolomide (TMZ) has been recently proposed as a treatment for gliomatosis cerebri (GC), but no predictive or prognostic markers have been identified so far. Because 1p19q codeletion and methylguanine methyl transferase promoter (MGMTP) methylation have been correlated with chemosensitivity of gliomas, their value was investigated in a cohort of patients with GC treated with TMZ. METHODS: A cohort of 25 GC patients who were treated with TMZ was investigated for 1p19q codeletion and O6-methylguanine DNA. RESULTS: Patients with a 1p/19q codeletion had a higher response rate (88% [8/9] vs 25% [4/16], p = 0.002), higher progression-free survival (24.5 vs 13.7 months, p = 0.017), and higher overall survival (66.8 vs 15.2 months, p = 0.011) than patients without 1p/19q codeletion. Fourteen of 19 evaluable tumors for MGMTP status were methylated. MGMTP methylation was associated with 1p/19q codeletion (p = 0.045). Patients with unmethylated MGMTP tended to have a shorter progression-free survival and a higher rate of progressive disease. CONCLUSION: Response rate to temozolomide and prognosis seem tightly correlated to 1p19q loss. The impact of methylguanine methyl transferase promoter methylation status on gliomatosis cerebri is still unsettled in this population.

Polymorphism in Sp1 recognition site of the EGF receptor gene promoter and risk of glioblastoma.

We investigated two polymorphisms of the epidermal growth factor receptor promoter as potential risk factors and prognostic markers for glioblastoma. The -216T allele (which results in a 30% higher activity) was more frequent in the patients compared with the control population (224/376 = 59.6% vs 165/352 = 46.8%; p = 0.0006) corresponding to an odd ratio of 1.67 (1.24; 2.25). A modest difference in median survival was also associated with the TT genotype.

EGFR tyrosine kinase domain mutations in human gliomas.

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor effective in patients with lung cancer with mutations in exons 19 and 21 of the EGFR tyrosine kinase domain. In this study, the authors tested the presence of such mutations in 95 gliomas including glioblastomas, anaplastic oligodendrogliomas, and low-grade gliomas. No mutation was found, which suggests that the biology of EGFR in gliomas is different from lung cancer and that this may be a factor in the resistance of glioblastomas to gefitinib.

Correlations between molecular profile and radiologic pattern in oligodendroglial tumors.

OBJECTIVE: To investigate possible correlations between tumor location and genetic alterations in a series of oligodendrogliomas. METHODS: A series of 158 consecutive oligodendrogliomas were retrospectively reviewed. In each case, the radiologic picture and the chromosome 1p (chr 1p) status of the tumor detected by the loss of heterozygosity technique were analyzed. Correlation between tumor location and molecular profile was made by chi2 tests. RESULTS: Eighty-eight of the 158 patients had low-grade oligodendrogliomas, and 70 had anaplastic oligodendrogliomas. Overall, oligodendrogliomas with chr 1p loss were located preferentially in the anterior part of the brain, whereas tumors with intact chr 1p affected mainly the posterior part of the brain (p = 0.0038). In terms of lobar involvement, a preferential location of oligodendrogliomas with chr 1p loss was found in the frontal lobes as compared with the temporal, parietal, and occipital tumors (p < 0.01). CONCLUSION: There is a significant correlation between loss of heterozygosity on chromosome 1p and tumor location in oligodendrogliomas, suggesting that subtypes of oligodendrogliomas could derive from site-specific precursors.

Molecular analysis of apo(a) fragmentation in polygenic hypercholesterolemia: characterization of a new plasma fragment pattern.

Hypercholesterolemia is frequently associated with elevated Lp(a) levels, an independent risk factor for coronary, cerebrovascular, and peripheral vascular disease. A portion of apolipoprotein(a) [apo(a)] circulates as a series of fragments derived from the N-terminal region of apo(a). The relationship of elevated lipoprotein(a) [Lp(a)] levels to those of circulating apo(a) fragments in polygenic hypercholesterolemia is indeterminate. Therefore, plasma Lp(a) and plasma and urinary apo(a) fragment levels were measured by ELISA in 82 patients with polygenic type IIa hypercholesterolemia (low density lipoprotein cholesterol >/=4.13 mmol/L and triglycerides <2.24 mmol/L) and in 90 normolipidemic subjects. Lp(a) levels were significantly elevated in patients compared with control subjects (0.35+/-0.4 and 0.24+/-0.31 mg/mL, respectively; median 0.13 and 0.11 mg/mL, respectively; P=0.039), although apo(a) isoform distribution did not differ. Patients displayed significantly higher plasma and urinary apo(a) fragment levels than did control subjects (respective values were as follows: 4.97+/-5.51 and 2.15+/-2.57 [median 2.85 and 1.17] microg/mL in plasma, P<0.0001; 75+/-86 and 40+/-57 [median 38 and 17] ng/mg urinary creatinine in urine, P<0.0001). The ratio of plasma apo(a) fragments to Lp(a) levels was also significantly higher in patients than in control subjects (1.93+/-1.5% and 1.75+/-2.36%, respectively; P<0.0001). We conclude that increased plasma Lp(a) levels in polygenic hypercholesterolemia are associated with elevated circulating levels of apo(a) fragments but that this increase is not due to decreased renal clearance of apo(a) fragments. Furthermore, we identified a new pattern of apo(a) fragmentation characterized by the predominance of a fragment band whose size was related to that of the parent apo(a) isoform and that was superimposed on the series of fragments described previously by Mooser et al (J Clin Invest. 1996; 98:2414-2424). This new pattern was associated with small apo(a) isoforms and did not discriminate between hypercholesterolemic and normal subjects. However, this new apo(a) fragment pattern may constitute a novel marker for cardiovascular risk.

Structural elucidation of the N- and O-glycans of human apolipoprotein(a): role of o-glycans in conferring protease resistance.

Apolipoprotein(a) (apo(a)) is a multikringle domain glycoprotein that exists covalently linked to apolipoprotein B100 of low density lipoprotein, to form the lipoprotein(a) (Lp(a)) particle, or as proteolytic fragments. Elevated plasma concentrations of apo(a) and its fragments may promote atherosclerosis, but the underlying mechanisms are incompletely understood. The factors influencing apo(a) proteolysis are also uncertain. Here we have used exoglycosidase digestion and mass spectrometry to sequence the Asn (N)-linked and Ser/Thr (O)-linked oligosaccharides of human apo(a). We also assessed the potential role of apo(a) O-glycans in protecting thermolysin-sensitive regions of the polypeptide. Apo(a) contained two major N-glycans that accounted for 17% of the total oligosaccharide structures. The N-glycans were complex biantennary structures present in either a mono- or disialylated state. The O-glycans were mostly (80%) represented by the monosialylated core type 1 structure, NeuNAcalpha2-3Galbeta1-3GalNAc, with smaller amounts of disialylated and non-sialylated O-glycans also detected. Removal of apo(a) O-glycans by sialidase and O-glycosidase treatment dramatically increased the sensitivity of the polypeptide to thermolysin digestion. These studies provide the first direct sequencing data for apo(a) glycans and indicate a novel function for apo(a) O-glycans that is potentially related to the atherogenicity of Lp(a).

Functional analysis of the chimpanzee and human apo(a) promoter sequences: identification of sequence variations responsible for elevated transcriptional activity in chimpanzee.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

Sequence polymorphisms in the apolipoprotein(a) gene and their association with lipoprotein(a) levels and myocardial infarction. The ECTIM Study.

Lp(a) concentrations are largely determined by apo(a) isoform size, but several studies have shown that apo(a) isoforms could not entirely explain the increase of Lp(a) levels observed in patients with coronary heart disease (CHD). Since up to 90% of the variance in Lp(a) levels has been suggested to be attributable to the apo(a) locus, the hypothesis that polymorphisms of the apo(a) gene other than size could contribute to the increase of Lp(a) levels in CHD patients must be considered. This hypothesis was tested in the ECTIM Study comparing 594 patients with myocardial infarction and 682 control subjects in Northern Ireland and France. In addition to apo(a) phenotyping, five previously described polymorphisms of the apo(a) gene were genotyped: a (TTTTA)n repeat at position -1400 from the ATG, a G/A at -914, a C/T at -49, a G/A at -21 and a Met/Thr affecting amino acid 4168. As reported earlier [Parra HJ, Evans AE, Cambou JP, Amouyel P, Bingham A, McMaster D, Schaffer P, Douste-Blazy P, Luc G, Richard JL, Ducimetiere P, Fruchart JC, Cambien F. A case-control study of lipoprotein particles in two populations at contrasting risk for coronary heart disease. The ECTIM study. Arterioscler Thromb 1992; 12:701-707], mean Lp(a) levels were higher in cases than in controls (20.7 vs 14.6 mg/dl in Belfast, 17.2 vs 8.9 mg/dl in France, P < 0.001 for case-control and population differences). In the present study, mean apo(a) isoform size differed significantly between cases and controls (25.7 vs 26.6 kr in Belfast, 25.9 vs 27.4 kr in France, P < 0.001 for case-control and P = 0.13 for population difference). After adjustment for apo(a) isoforms, Lp(a) levels remained significantly higher in cases than in controls (difference, 4.6 mg/dl; P < 0.001). Genotype and allele frequencies did not differ significantly between cases and controls for any of the five polymorphisms studied. The five polymorphisms were in strong linkage disequilibrium and had a combined heterozygosity of 0.83. In multivariate regression analysis adjusted for apo(a) isoforms, only the (TTTTA)n polymorphism was significantly associated with Lp(a) levels; it explained 4.5% of Lp(a) variability in cases and 3.1% in controls. The Lp(a) case/control difference was not reduced after taking into account the (TTTTA)n effect. We conclude that the increase of Lp(a) levels observed in MI cases, and which was not directly attributable to apo(a) size variation, was not related to the five polymorphisms of the apo(a) gene considered.

Sequence diversity in 36 candidate genes for cardiovascular disorders.

Two strategies involving whole-genome association studies have been proposed for the identification of genes involved in complex diseases. The first one seeks to characterize all common variants of human genes and to test their association with disease. The second one seeks to develop dense maps of single-nucleotide polymorphisms (SNPs) and to detect susceptibility genes through linkage disequilibrium. We performed a molecular screening of the coding and/or flanking regions of 36 candidate genes for cardiovascular diseases. All polymorphisms identified by this screening were further genotyped in 750 subjects of European descent. In the whole set of genes, the lengths explored spanned 53.8 kb in the 5' regions, 68.4 kb in exonic regions, and 13 kb in the 3' regions. The strength of linkage disequilibrium within candidate regions suggests that genomewide maps of SNPs might be efficient ways to identify new disease-susceptibility genes, provided that the maps are sufficiently dense. However, the relatively large number of polymorphisms within coding and regulatory regions of candidate genes raises the possibility that several of them might be functional and that the pattern of genotype-phenotype association might be more complex than initially envisaged, as actually has been observed in some well-characterized genes. These results argue in favor of both genomewide association studies and detailed studies of the overall sequence variation of candidate genes, as complementary approaches.

Molecular cloning of the cDNA encoding the carboxy-terminal domain of chimpanzee apolipoprotein(a): an Asp57 --> Asn mutation in kringle IV-10 is associated with poor fibrin binding.

Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.

Chimpanzee lipoprotein(A): Relationship between apolipoprotein(A) isoform size and the density profile of lipoprotein(A) in animals with different heterozygous apo(A) phenotypes.

In a previous study [C. Doucet et al., J. Lipid Res 35:263-270, 1994], we have shown that plasma lipoprotein (a) [Lp(a)] levels were significantly elevated in a population of unrelated chimpanzees as compared to those in normolipidemic human subjects. Nonetheless, the inverse correlation between Lp(a) levels and apolipoprotein (a) [apo(a)] isoforms typical of man was maintained in the chimpanzee. In the present study, we describe the density profiles of apo B- and apo A1-containing lipoproteins and of Lp(a) in chimpanzee plasmas heterozygous for apo(a) isoforms after fractionation by single spin ultracentrifugation in an isopycnic gradient. The distribution of apo(a) isoforms in the density gradient was also examined by SDS-agarose gel electrophoresis and immunoblotting using chemiluminescence detection. In all double-band phenotypes examined, the smallest isoform was present along the entire length of the density gradient. The density distribution of the second isoform varied according to the size difference between the respective isoforms. Two isoforms close in size (difference in apparent molecular mass = 60 kDa) were present together in every gradient subfraction. On the contrary, when the two isoforms displayed distinct molecular mass (maximal difference in apparent molecular mass = 340 kDa), then the largest was principally present in the densest fractions of the gradient (d > 1.1 mg/ml). These observations suggest that Lp(a) particles with small apo(a) isoforms are more susceptible to interact with other lipoproteins than are Lp(a) particles with large isoforms.

Elevated lipoprotein(a) levels and small apo(a) isoforms are compatible with longevity: evidence from a large population of French centenarians.

Epidemiological studies have shown lipoprotein(a) (Lp(a)) to be an independent risk factor for cardiovascular disease. Lp(a) is a cholesterol-rich, low-density lipoprotein (LDL)-like particle to which a large glycoprotein, apolipoprotein(a) (apo(a)) is attached. Plasma Lp(a) levels are highly genetically determined and influenced to a minor degree by environmental factors. In an effort to determine whether Lp(a) might be associated with longevity, we have evaluated Lp(a) levels and apo(a) isoform sizes in a population of French centenarians (n = 109) compared to a control group (n = 227). The mean age of centenarians was 101.5 +/- 2.4 years while the control group was 39.4 +/- 7.2 years. Plasma levels of total cholesterol and triglyceride were within the normal range in both centenarian and control subjects. Lp(a) levels were higher in centenarians (both male and female) than in the normolipidemic control group (mean Lp(a) level = 0.33 +/- 0.42 and 0.22 +/- 0.27 mg/ml, respectively, P < 0.005). The distribution of apo(a) isoforms was significantly shifted towards small isoform size in the centenarian population as compared to the controls (54.4 and 41.4% of isoforms < or = 27 kringles (kr), respectively, P = 0.04). Nonetheless, the apo(a) size distribution in centenarians did not entirely explain the high Lp(a) levels observed in this population. Factors other than apo(a) size, and which may be either genetic or environmental in nature, appear to contribute to the elevated plasma Lp(a) levels of our centenarian population. We conclude therefore that high plasma Lp(a) levels are compatible with longevity.

Lipoprotein(a) is a potent chemoattractant for human peripheral monocytes.

We have previously reported that the serine protease plasmin triggers chemotaxis in human peripheral monocytes, but not in polymorphonuclear leukocyte. We now show that the structurally related lipoprotein(a) (Lp[a]) as well as recombinant apolipoprotein(a) (apo[a]) trigger chemotactic responses in human monocytes equipotent to that observed with the standard chemoattractant FMLP. The chemotactic effects of Lp(a) and FMLP were additive. Low density lipoprotein (LDL) did not elicit any significant chemotactic response nor did it interfere with that triggered by Lp(a). As assessed by checkerboard analysis, Lp(a)-mediated monocyte locomotion was a true chemotaxis. Both plasminogen as well as catalytically inactivated plasmin inhibited monocyte migration elicited by Lp(a), suggesting binding of Lp(a) to plasminogen binding sites. Lp(a)-mediated signaling proceeds through a pertussis toxin-sensitive guanosine triphosphate (GTP)-binding protein and activation of protein kinase C as implicated by the effects of 1-O-hexadecyl-2-O-methyl-rac-glycerol and chelerythrine. Lp(a) induced generation of guanosine 3',5'-cyclic monophosphate (cGMP), apparently crucial for the Lp(a)-mediated chemotaxis, because an inhibitor of soluble guanylyl cyclase, LY83583, reduced both the Lp(a)-induced cGMP formation as well as the monocyte migration. The latter effect of LY83583 was antagonized by the stable cGMP analog 8-pCPT-cGMP. The data indicate that Lp(a) triggers chemotaxis in human monocytes by way of a cGMP-dependent mechanism. Our findings may have important implications for the atherogenesis associated with elevated levels of Lp(a).

Pathophysiological implication of the structural domains of lipoprotein(a).

Numerous epidemiological studies have shown that lipoprotein(a) (Lp(a)) is an independent risk factor for the premature development of cardiovascular disease. In spite of such evidence, the structural and functional features of this atherogenic, cholesterol-rich particle are not clearly understood. We have demonstrated the presence of two distinct structural domains in apolipoprotein(a) (apo(a)), which are linked by a flexible and accessible region located between kringles 4-4 and 4-5. We have isolated the Lp(a) particle following removal of the N-terminal domain by proteolytic cleavage; the residual particle, containing the C-terminal domain (comprising the region from Kr 4-5 to the protease domain), is linked to apo B-100 by disulphide linkage, and is termed 'mini-Lp(a)'. Mini-Lp(a) exhibited the same binding affinity to fibrin as the corresponding Lp(a). This finding indicated that the kringles responsible for fibrin binding are restricted to Kr 4-5 to Kr 4-10, an observation consistent with the failure of the N-terminal domain to bind to fibrin. N-terminal fragments of apo(a) have been detected in the urine of normal subjects, thereby indicating that part of the catabolism of Lp(a), which is largely indeterminate, could occur via the renal route.