Clinical and molecular characteristics and treatment outcomes of advanced right-colon, left-colon and rectal cancers: data from 1180 patients in a phase III trial of panitumumab with an extended biomarker panel.

BACKGROUND: Primary tumour location (PTL) is being adopted by clinicians to guide treatment decisions in metastatic colorectal cancer (mCRC). Here we test PTL as a predictive marker for panitumumab efficacy, and examine its relationship with an extended biomarker profile. We also examine rectal tumours as a separate location. PATIENTS AND METHODS: mCRC patients from the second-line PICCOLO trial of irinotecan versus irinotecan/panitumumab (IrPan). PTL was classified as right-PTL, left-PTL or rectal-PTL. PTL was assessed as a predictive biomarker for IrPan effect in RAS-wild-type (RAS-wt) patients (compared with irinotecan alone), then tested for independence alongside an extended biomarker profile (BRAF, epiregulin/amphiregulin (EREG/AREG) and HER3 mRNA expression). RESULTS: PTL data were available for 1180 patients (98.5%), of whom 558 were RAS-wt. High HER3 expression was independently predictive of panitumumab overall survival improvement, but PTL and EREG/AREG were not. IrPan progression-free survival (PFS) improvement compared with irinotecan was seen in left-PTL [hazard ratio (HR) = 0.61, P = 0.002) but not right-PTL (HR = 0.98, P = 0.90) (interaction P = 0.05; RAS/BRAF-wt interaction P = 0.10), or in rectal-PTL (HR = 0.82, P = 0.20) (interaction P = 0.14 compared with left-PTL; RAS/BRAF-wt interaction P = 0.04). Patients with right-PTL and high EREG/AREG or HER3 expression, had IrPan PFS improvement (high EREG/AREG HR = 0.20, P = 0.04; high HER3 HR = 0.33, P = 0.10) compared with irinotecan. Similar effect was seen for rectal-PTL patients (high EREG/AREG HR = 0.44, P = 0.03; high HER3 HR = 0.34, P = 0.05). CONCLUSIONS: RAS-wt patients with left-PTL are more likely to have panitumumab PFS advantage than those with right-PTL or rectal-PTL. However, an extended biomarker panel demonstrated significant heterogeneity in panitumumab PFS effect within a tumour location. AREG/EREG and HER3 mRNA expression identifies patients with right-PTL or rectal-PTL who achieve similar PFS effect with panitumumab as left-colon patients. Testing could provide a more reliable basis for clinical decision making. Further validation and development of these biomarkers is required to optimise routine patient care. CLINICAL TRIAL REGISTRATION: ISRCTN identifier: ISRCTN93248876.

The NOTCH4 locus is associated with susceptibility to schizophrenia.

Linkage disequilibrium mapping of the MHC region in 80 British parent-offspring trios showed that NOTCH4 was highly associated with schizophrenia. The A-->G substitution in the promoter region and the (CTG)n repeat in exon 1 of NOTCH4 may be candidate sites conferring susceptibility to schizophrenia.

KRAS and BRAF mutations in Nigerian colorectal cancers.

PURPOSE: Activation of the KRAS oncogene is implicated in colorectal carcinogenesis and mutations have been reported in 30-50% of cases. BRAF mutation, though less common, is also reported and importantly associated with shorter progression-free interval. This study aims to determine the KRAS and BRAF mutation statuses of Nigerian colorectal cancers (CRC). METHODS: Mutation analysis was carried out on archival paraffin-embedded blocks of CRC tissues. KRAS codons 12, 13 and 61 and BRAF V600E were assessed by pyrosequencing after DNA extraction from 200 cases at the Leeds Institute of Molecular Medicine, St. James's University Hospital, UK. Mutation rates and the spectra were determined. RESULTS: Pyrosequencing was successful in 112 of 200 cases. KRAS mutation in codons 12 and 13 was demonstrated in 23 of 112 cases (21%); none in codon 61. BRAF mutation in codon 600 was demonstrated in 4.5%. CONCLUSION: This study shows that 21% of Nigerian CRC patients carry a KRAS mutation; half the rate in Caucasians; and that BRAF mutation also occurs in Nigerian CRC cancers.

Is the polymorphic microsatellite repeat of the dopamine beta-hydroxylase gene associated with biochemical variability of the catecholamine pathway in schizophrenia?

Six allelic fragments were typed by a polymerase chain reaction process with a pair of primers specific for a sequence containing the polymorphic (GT)n repeat, a microsatellite repeat, in the human dopamine beta-hydroxylase (DBH) gene. Their frequencies in unrelated patients with schizophrenia were 0.003 (A1), 0.114 (A2), 0.343 (A3), 0.526 (A4), 0.006 (A5), and 0.009 (A6), and in unrelated control subjects, 0.012 (A1), 0.086 (A2), 0.309 (A3), 0.574 (A4), 0.006 (A5), and 0.012 (A6). Kruskal-Wallis analysis revealed significant differences among the three groups, the drug-free and drug-treated patients, and the control subjects, in serum DBH activity of the subjects whose genotype was A2/A3 (H = 6.0, p < .05) or A3/A3 (H = 9.8, p < .01), in serum homovanillic acid concentration of those whose genotype was A3/A4 (H = 7.7, p < .025), and in serum tyrosine concentration of those whose genotype was A4/A4 (H = 8.3, p < .02). Mann-Whitney U test showed that in the subjects carrying the A3/A4 genotype, serum noradrenaline concentration of drug-treated patients was significantly higher than that of control subjects (N = 58, p < .02). These results suggest that genotypic polymorphism of the human DBH is likely to be associated with biochemical variability of the catecholamine pathway in schizophrenia.

Searching for a locus for schizophrenia within chromosome Xp11.

Three gene-rich loci-HS212G6, HSU93305, and HS884M20-within the short arm of the X chromosome have been examined for allelic association with schizophrenia by the transmission disequilibrium test in 70 families of male individuals affected with schizophrenia. Neither the HS212G6 nor HS884M20 was found to be associated with schizophrenia. The HSU93305 locus, however, was significantly associated with schizophrenia (X(2)=17.92, df=3, P<0.001). The HSU93305 locus contains four distinct genes. They code, respectively, for A4 differentiation-dependent protein, triple LIM domain protein, synaptophysin, and calcium channel alpha-1 subunit. It is possible that one of these genes or some loci near to it may predispose a vulnerability to schizophrenia. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:4-7, 2000.

Studies on neurochemical heterogeneity in healthy parents of schizophrenic patients.

Serum homovanillic acid (HVA), norepinephrine (NE), phenylalanine (Phe) and tyrosine (Tyr) have been examined in 80 healthy parents of schizophrenic patients and 26 normal control subject. Analysis of variance revealed a significant difference in serum HVA concentration among the three groups: the parents whose ill offspring became fairly well after neuroleptic treatment for more than three months (n = 33), those whose offspring were still actively ill after neuroleptic treatment (n = 33), and normal control subjects (F = 3.98, df = 2, 89, p < 0.05). The t-test showed that serum HVA was significantly higher in the parents whose ill offspring became fairly well after neuroleptic treatment (11.8 +/- 5.0 ng/ml) than in normal control subjects (8.7 +/- 3.5 ng/ml, p < 0.01), but was not significantly higher in the parents whose offspring were still actively ill (10.5 +/- 3.7 ng/ml, p > 0.05). There was a significant difference between the serum NE concentrations of the parents of female patients (515 +/- 224 pg/ml, n = 21) and those of male patients (401 +/- 186 pg/ml, n = 55, p < 0.05). No significant differences were found in the serum concentrations of Phe and Tyr. These results suggest that there may be neurochemical heterogeneity in the parents of schizophrenic patients, which may be involved in the response of schizophrenic offspring to neuroleptic treatment and in the gender differences of schizophrenia.

Studies on concentrations of NA and HVA and activity of DBH in the serum from schizophrenic patients, first-degree relatives and normal subjects.

The serum noradrenaline (NA), homovanillic acid (HVA) and dopamine beta-hydroxylase (DBH) have been examined in neuroleptic-free and -treated patients, healthy first-degree relatives of the patients and normal subjects. Analysis of variance (ANOVA) revealed significant differences in the concentrations of serum NA(F = 2.91, p < 0.05) and HVA (F = 3.58, p < 0.05), and in the activity of serum DBH (F = 2.77, p < 0.05) among the four groups. The serum NA was significantly higher in neuroleptic-free patients (475 +/- 220 pg/ml, n = 18), than in healthy first-degree relatives (343 +/- 189 pg/ml, n = 37, p < 0.05) or in normal subjects (354 +/- 111 pg/ml, n = 17, p < 0.05), and it also was significantly higher in neuroleptic-treated patients (442 +/- 223 pg/ml, n = 58) than in healthy first-degree relatives (p < 0.05) or in normal subjects (p < 0.05). There was a trend towards high serum HVA in neuroleptic-free patients (11.3 +/- 6.3 ng/ml, n = 17) compared with the other three groups. The serum DBH activity was high in neuroleptic-free patients (31.2 +/- 15.6 nmol/min/ml, n = 17), and significantly in comparison with those treated with neuroleptic drugs (21.6 +/- 10.9 nmol/min/ml, n = 56, p < 0.05). There was a significant negative correlation between HVA concentration and DBH activity in the serum from neuroleptic-free patients (r = -0.64, n = 16, p < 0.01), and there appeared to be three subgroups with alterations of serum DBH activity and HVA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of the relationship between the DBH activity in serum and a MspI polymorphic site in intron 9 of the human DBH gene in schizophrenia.

Intron 9 of the human dopamine beta-hydroxylase (DBH) gene was amplified using a long PCR procedure in unrelated patients with schizophrenia and unrelated control subjects. MspI digestion of the PCR fragments showed a two allele polymorphism. A1 and A2 Kruskal-Wallis analysis revealed a significant difference in serum DBH activity among the three groups carrying the A1/A2 genotype, the drug-free and drug treated patients, and the control subjects (H = 12.2, df = 2, p < 0.005), and also a significant difference among the three subgroups of drug-treated patients carrying the genotype of A1/A1, A2/A2 or A1/A2 (H = 10.4, df = 2, p < 0.01). The present results suggest that the MspI polymorphic site in intron 9 of the human DBH gene may be associated with alterations of DBH activity in schizophrenia and with the influence of neuroleptic drugs on the DBH activity as well.

Comparisons of morphology and growth rate between cells from skin biopsies of schizophrenic patients and those from normal individuals.

Skin biopsies have been used to establish cell cultures from ten schizophrenic individuals and ten controls. Studies of the time of outgrowth of different cell types from the tissue fragments and the subsequent growth rates and morphology showed no difference between the primary cultures from the schizophrenic patients and controls. The primary cultures were trypsinized and grown for three passages before storing in liquid nitrogen. No difference between cell strains established from schizophrenic skin donors and cell strains from healthy controls was noted in this phase of growth. Aliquots of cells were thawed out from liquid nitrogen, grown for one passage and then the cell aggregation rate was tested. No difference was found between cells from schizophrenic skin donors and cells from controls. Differences between the two cell strains established from certain individuals did occur, suggesting that morphology and growth rates should be noted before cell strains are used for further studies.

Low concentrations of serum tyrosine in neuroleptic-free schizophrenics with an early onset.

The concentrations of serum tyrosine, phenylalanine and tryptophan have been determined in 23 male neuroleptic-free patients with schizophrenia and 28 male healthy control subjects. Tyrosine was significantly lower in neuroleptic-free patients with an early onset (starting before adulthood) than in healthy control subjects (p < 0.05), and the ratio of tyrosine to phenylalanine was significantly lower in neuroleptic-free patients with an early onset than in those with a late onset (starting at adulthood). No significant differences in these three amino acids were found between the patients with a late onset and healthy control subjects. The present findings suggest that there may be a disturbance of balance between tyrosine and phenylalanine in early-onset patients with schizophrenia.

A study of enzymes involved in catecholamine metabolism in parents of patients with schizophrenia.

The concentrations of serum homovanillic acid (HVA), norepinephrine (NE), tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp), and the activities of serum dopamine-beta-hydroxylase (DBH), platelet monoamine oxidase (MAO), and erythrocyte catechol-O-methyl transferase (COMT) were measured in 68 healthy parents who had schizophrenic offspring. The results show a significant correlation between the parents of schizophrenic patients in serum HVA (r=0.38, n=34, p<0.05), NE (r=0.40, n=33, p<0.02), Phe (r=0.44, n=34, p<0.0l), Tyr (r=0.43, n = 34, p <0.02) and DBH activity (r=0.51, n = 30, p <0.005), but do not show a significant correlation in erythrocyte COMT (r=0.01, n=27), platelet MAO (r=0.04, n=23) or serum Trp (r=0.10, n=34). There were no significant correlations in these measurements between randomly matched parents. The present study suggests that both parental sides of schizophrenic patients are likely to have similar alleles associated with the catecholamine pathway, and their ill offspring may possess a double dose of the schizophrenogenic alleles.

A study of linkage disequilibrium between polymorphic loci for monamine oxidases A and B in schizophrenia.

Two X-linked microsatellites, (AC) n repeats at the monoamine oxidase (MAO)-A locus and (TG)n repeats at the MAO-B locus, were studied in 140 unrelated Caucasian male patients with schizophrenia and 91 unrelated Caucasian male controls. Among these subjects, we totally typed out nine alleles for the (AC) n repeats and eight alleles for the (TG) n repeats by using a PCR-based procedure. Allelic frequencies of either (AC) n repeats or (TG) n repeats were not found to be significantly different between patients and controls. However, a significant excess of the (AC)18/(TG)23 haplotype with a relative risk of 4.05 (95%; CI 1.15-14.26) was observed in patients with schizophrenia (Fisher's P = 0.011). The coefficient of linkage disequilibrium (delta) for the (AC)18/(TG)23 haplotype was 0.019 in schizophrenic patients and -0.046 in control subjects, respectively. The latter reached statistical significance (chi 2 = 6.02; df = 1; P < 0.02). The present findings suggest that linkage disequilibrium between polymorphic loci for human MAO-A and MAO-B may be associated with schizophrenia, and the (AC)18/(TG)23 haplotype may render an individual more vulnerable to such an illness.

Lack of evidence for association between the COMT locus and schizophrenia.

Family-based studies have been conducted with restriction fragment length polymorphism (RFLP) analysis for testing association between polymorphisms for the catechol-O-methyltransferase (COMT) locus and schizophrenia in 49 Caucasian nuclear families consisting of fathers, mothers and offspring affected with schizophrenia. The present results did not support the hypothesis that the COMT gene might play an important role in predisposing an individual to a genetic risk for schizophrenia. Neither did we find a significant association of the COMT locus with violent behaviour in schizophrenia. Nevertheless, there may be a susceptibility gene for schizophrenia in a distinct region from the COMT locus on chromosome 22q, as a genome scan has suggested recently.

Association of polymorphic VNTR region in the first intron of the human TH gene with disturbances of the catecholamine pathway in schizophrenia.

In the present study, five allelic fragments were typed by a polymerase chain reaction (PCR) process with a pair of primers specific for the tetranucleotide (TCAT) repeat sequence in the first intron of the human tyrosine hydroxylase (TH) gene and their sizes (bp) were 114 (A), 118 (B), 122 (C), 126 (D) and 130 (E), respectively. The AE genotypic frequency was found to be significantly higher in unrelated patients with schizophrenia than in unrelated control subjects (chi 2 = 4.18, p < 0.05). ANOVA revealed a significant difference between the three groups (neuroleptic-free patients possessing or not possessing the AE genotype, and unrelated control subjects) in the concentration of serum noradrenaline (F = 4.96, df = 2.79, p < 0.01), but no significant differences were found between the three groups in the concentrations of serum homovanillic acid, phenylalanine and tyrosine. These results suggest that the polymorphic intron 1 of the human TH gene may be associated with disturbances of the catecholamine pathway in schizophrenia.

TaqI polymorphic sites at the human dopamine beta-hydroxylase gene possibly associated with biochemical alterations of the catecholamine pathway in schizophrenia.

Two parts of the dopamine beta-hydroxylase (DBH) gene, one a 7.5-kb single copy fragment (F1) spanning the 5'-flanking region to exon 3 and the second a 9.0-kb single copy fragment (F2) spanning exon 3 to exon 7, were amplified by a long PCR procedure in 161 unrelated patients with schizophrenia and 67 unrelated control subjects. The PCR products were completely digested with the restriction enzyme TaqI. These subjects were classified into genetic subgroups according to the TaqI restriction fragment length polymorphisms (RFLPs) for the human DBH gene, and the association of the TaqI RFLPs with biochemical alterations of the catecholamine pathway in schizophrenia was then examined. The frequencies of the two TaqI polymorphic sites did not show significant differences between the patients and control subjects, but the TaqI RFLPs were found to be associated with biochemical alterations of the catecholamine pathway in schizophrenia.

A study of a genetic association between the PTGS2/PLA2G4A locus and schizophrenia.

Six single nucleotide polymorphisms (SNPs) present in the PTGS2/PLA2G4A locus were detected among 118 British family trios of schizophrenia patients. The transmission disequilibrium test showed that SNP4 located in the 5'-flanking region of the PLA2G4A gene was associated with schizophrenia and that the haplotype analysis also showed a genetic association between the PTGS2 gene and schizophrenia. Because these two genes are arranged in a head-to-head configuration and separated by just about 149kb of DNA, they may have a combined effect on susceptibility to schizophrenia in some cases.

Decreased tyrosine transport in fibroblasts from schizophrenics: implications for membrane pathology.

Two independent studies reported recently have shown a significant decrease in Vmax of tyrosine transport in fibroblasts grown from schizophrenics' skin compared with controls. It has also been shown that tyrosine transport into the brain is decreased in schizophrenics compared with controls. In view of the importance of these findings in elucidating the biochemical mechanism(s) associated with schizophrenia, we have studied the kinetics of tyrosine transport and the levels of monoamine oxidase (MAO) activity in fibroblasts grown from the skins of schizophrenics and unrelated control subjects. Using the Lineweaver-Burk plot, the Eadie Hostee plot and the Hanes plot we have calculated the Km and Vmax for tyrosine transport. We have found a significant decrease in the Km and Vmax values for tyrosine transport in schizophrenics compared with control fibroblast samples. No changes were observed in the levels of MAO. Using Lineweaver-Burk plot (1/S Versus 1/V) it has been shown that the tyrosine transport inhibition is uncompetitive. This finding proposes that the inhibition is in the substrate transport protein complex, which may be taking place during the transit of the substrate through the cell membrane. From the observed findings and from the literature evidence we suggest that the altered metabolism of phospholipids in schizophrenics, such as deficiency of arachidonic acid and docosahexaenoic acid, may be contributing to this observed phenomena.

Possible association of catecholamine turnover with the polymorphic (TCAT)n repeat in the first intron of the human tyrosine hydroxylase gene.

Five allelic fragments were typed by a PCR-based process with a pair of primers specific for the polymorphic sequence due to (TCAT)n tetranucleotide repeat, a microsatellite repeat, in the first intron of the human tyrosine hydroxylase gene, i.e. A1, A2, A3, A4 and A5. Comparisons of some neurochemical parameters of the catecholamine pathway were then made between the unrelated individuals genotypically classified into six subgroups, five homozygotic and one heterozygotic. Among the six subgroups, the individuals with the A2/A2 genotype had the highest levels of serum noradrenaline and those with the A4/A4 genotype had the lowest, and the individuals with the A1/A1 genotype had the highest levels of serum homovanillic acid. These findings suggest that polymorphism of the (TCAT)n repeat in the first intron of the human tyrosine hydroxylase gene may be associated with catecholamine turnover. Possibly, the two alleles, A2 and A4, may be related to the high and low excitabilities of the noradrenergic nerves, respectively, and the allele, A1, may be associated with the up-regulation of dopamine turnover.

Increased serum IgE in schizophrenic patients who responded poorly to neuroleptic treatment.

Total serum IgE and plasma Interleukin (IL)-4 have been determined in patients with schizophrenia, 26 neuroleptic-free, and 81 neuroleptic-treated, and in 46 healthy control subjects. The total serum IgE was significantly higher in the patients who responded poorly to neuroleptic treatment compared with the other four groups (F = 4.27, df = 4, 148, P < 0.003). No significant changes were found in plasma IL-4 levels between any of the five groups. It is possible that raised serum IgE levels may characterise a subgroup of schizophrenia.

Increase of plasma IL-6 concentration with age in healthy subjects.

The present study demonstrated that plasma IL-6 concentration was higher in older subjects than in younger ones and significantly in the male group (P = 0.02); Spearman rank correlation showed that plasma IL-6 concentration was positively correlated with age (r = 0.28, N = 55, P < 0.05); there was a highly significant correlation between the concentrations in plasma IL-6 and IL-1 alpha (r = 0.51, N = 52, P < 0.001). These findings suggest the possibility that age-related changes of plasma IL-6 and IL-1 alpha may provide a pathological basis for the susceptibility to such illness as commonly occurs in elderly people, especially Alzheimer's disease as the two interleukins can induce the production of alpha 1-antichymotrypsin and beta-amyloid protein precursor.