Current use of pharmacogenetic testing: a national survey of thiopurine methyltransferase testing prior to azathioprine prescription.

BACKGROUND: Azathioprine is an immunosuppressant prescribed for the treatment of inflammatory conditions and after organ transplantation. Risk of neutropaenia has limited the effective use of azathioprine (AZA) and driven requirements for careful monitoring and blood tests. Thiopurine methyltransferase (TPMT) is a genetically moderated key enzyme involved in the metabolism of AZA that can be used to stratify individuals into different levels of risk of developing neutropaenia. Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). OBJECTIVE: To identify the current uptake of TPMT enzyme-level testing, TPMT genotype testing, and, the role of guidelines; to inform the prescribing and monitoring of AZA. METHOD: A survey was mailed to a consultant dermatologist, gastroenterologist, and rheumatologist at every NHS Hospital Trust in England. The survey comprised mainly closed questions exploring: use of AZA and monitoring; use of TPMT enzyme-level testing and genotype testing; and, the role of guidelines to guide prescribing practice. RESULTS: A 70% (n=287) response rate was obtained. The majority of respondents reported prescribing AZA (99%, n=283). Prescribing and monitoring patterns differed between individual respondents and between the three disciplines. TPMT enzyme-level testing was reportedly used by 67% (n=189) of respondents, but this differed by discipline (dermatologists 94%, gastroenterologists 60%, rheumatologists 47%). In 91% of cases enzyme-level testing was carried out prior to prescribing AZA. Genotype testing is not typically available to NHS clinicians but 15 clinicians (six dermatologists, six gastroenterologists, three rheumatologists) reported using it. Most consultants (82%) reported using guidelines to inform their AZA prescribing and monitoring (dermatologists 81%, gastroenterologists 75%, rheumatologists 94%). CONCLUSION: Two-thirds of the consultants surveyed in England are using TPMT enzyme-level testing, prior to AZA treatment. Uptake differs between specialities. High uptake of TPMT enzyme-level testing by dermatologists, compared with gastroenterologists and rheumatologists, may reflect national guidelines advocating its use prior to AZA. Uptake of enzyme-level testing may alter in other specialties as other guidelines are developed.

Patients with stable long-standing rheumatoid arthritis continue to deteriorate despite intensified treatment with traditional disease modifying anti-rheumatic drugs--results of the British Rheumatoid Outcome Study Group randomized controlled clinical trial.

OBJECTIVE: Patients with rheumatoid arthritis (RA) should start treatment early with the aim of suppressing the inflammatory process completely. It is not known if this strategy should, or can, be continued in later disease. METHODS: In a multicentre, randomized, observer-blinded, controlled trial, 466 patients with established RA (>5 yr), on stable therapy for at least 6 months, were randomized to adequate symptom control/shared care setting (SCSC) or aggressive treatment/hospital setting (ATH). All were reviewed annually by a rheumatologist. The primary outcome after 3 yr was the Health Assessment Questionnaire (HAQ). Others included the OMERACT core set and the Disease Activity Score (DAS) 28. RESULTS: Three hundred and ninety-nine patients completed the trial. There was a significant deterioration in HAQ in both arms. Only the physician global score differed between the arms. CONCLUSIONS: The trial showed no additional benefit of intensified treatment with traditional disease modifying anti-rheumatic drugs (DMARDs) in patients with stable, established RA. It proved hard to suppress C-reactive protein levels. Patients in the SCSC arm were able to initiate treatment changes when their symptoms deteriorated without frequent hospital assessment. Pending further evidence, the model of shared care with annual hospital review is as good as 4-monthly hospital review for these patients.

The British Rheumatoid Outcome Study Group (BROSG) randomised controlled trial to compare the effectiveness and cost-effectiveness of aggressive versus symptomatic therapy in established rheumatoid arthritis.

OBJECTIVES: To examine the effectiveness and cost-effectiveness of symptomatic versus aggressive treatment in patients with established, stable rheumatoid arthritis (RA). DESIGN: A randomised observer-blinded controlled trial and economic evaluation with an initial assessment at randomisation and follow-ups at 12, 24 and 36 months. SETTING: Five rheumatology centres in England. The 'symptomatic care' patients were managed predominantly in primary care with regular visits by a rheumatology specialist nurse. The 'aggressive care' patients were managed predominantly in the hospital setting. PARTICIPANTS: Patients with RA for more than 5 years were screened in rheumatology clinics. INTERVENTIONS: The symptomatic care patients were seen at home every 4 months by a rheumatology specialist nurse and annually by the rheumatologist. The aim of treatment was symptom control. The aggressive care patients were seen at least every 4 months in hospital. Their treatment was altered (following predefined algorithms) with the aim of suppressing both clinical and laboratory evidence of joint inflammation. MAIN OUTCOME MEASURES: The main outcome measure was the Health Assessment Questionnaire (HAQ). Others included the patient and physician global assessment, pain, tender and swollen joint counts, the erythrocyte sedimentation rate and the OSRA (Overall Status in Rheumatoid Arthritis) score. X-rays of the hands and feet were performed at the beginning and end of the study. The EQ-5D was used in the health economic evaluation. Comprehensive costs were also estimated and were combined with measures of outcome to examine between-group differences. RESULTS: A total of 466 patients were recruited; 399 patients completed the 3 years of follow-up. There was a significant deterioration in physical function (HAQ) in both arms. There was no significant difference between the groups for any of the clinical outcome measures except the physician global assessment [adjusted mean difference 3.76 (95% CI 0.03 to 7.52)] and the OSRA disease activity component [adjusted mean difference 0.41 (95% CI 0.01 to 0.71)], both in favour of the aggressive arm. During the trial, second-line drug treatment was changed in 77.1% of the aggressive arm and 59.0% of the symptomatic arm. There were instances when the rheumatologist should have changed treatment but did not do so, usually because of mild disease activity. The symptomatic arm was associated with higher costs and higher quality-adjusted life-years (QALYs). There was a net cost of 1517 Pounds Sterling per QALY gained for the symptomatic arm. Overall, the primary economic analysis and sensitivity analyses of the cost and QALY data indicate that symptomatic treatment is likely to be more cost-effective than aggressive treatment in 58-90% of cases. CONCLUSIONS: This trial showed no benefit of aggressive treatment in patients with stable established RA. However, it was difficult to persuade the rheumatologist and/or the patient to change treatment if the evidence of disease activity was minimal. Patients in the symptomatic arm were able to initiate changes of therapy when their symptoms deteriorated, without frequent hospital assessment. Approximately one-third of current clinic attenders with stable RA could be managed in a shared care setting with annual review by a rheumatologist and regular contact with a rheumatologist nurse. Further research is needed into disease progression and the use of biological agents, minimum disease activity level below which disease progression does not occur, cost-effectiveness through shared care modelling, the development of a robust and fail-safe system of primary-care based disease-modifying anti-rheumatic drug (DMARD) monitoring, and predicting response to DMARDs.

The detection of antibodies against Schistosoma mansoni soluble egg antigens (SEA) and CEF6 in ELISA, before and after chemotherapy.

Circulating IgG antibody reactivity and excreted egg counts were investigated in 489 Kenyans given chemotherapy for schistosomiasis mansoni. Antibody reactivity was measured in ELISA, using either unfractionated aqueous soluble constituents of Schistosoma mansoni eggs (SEA) or CEF6 (a soluble fraction of S. mansoni eggs containing two cationic antigens) as the antigen source. Antibody reactivity for each antigen source was strongly associated with egg counts, both pre- and post-treatment. Approximately 6 months after chemotherapy, egg counts were zero in 84% of the subjects. The mean optical densities (OD) measured in the post-treatment ELISA were 60% (CEF6) or 45% (SEA) lower than the pre-treatment values, the reduction in the OD with CEF6 as antigen source being significantly greater than that observed with SEA (P <0.001). The usefulness of an assay for antibody reactivity in monitoring the effects of the treatment of schistosomiasis is discussed.

A novel TP53 splicing mutation in a Li-Fraumeni syndrome family: a patient with Wilms' tumour is not a mutation carrier.

We report a Li-Fraumeni syndrome family in which we have detected a splice acceptor mutation in intron 3 of TP53. The mutation affects one of the invariant residues at the splice acceptor site, as a result of which two aberrant transcripts are produced. A child with Wilms' tumour aged 3 years in this family was shown not to be a mutation carrier.

Cancer phenotype correlates with constitutional TP53 genotype in families with the Li-Fraumeni syndrome.

The Li-Fraumeni cancer predisposition syndrome is associated with germline TP53 mutations in the majority of families. We have investigated cancer incidence in 34 Li-Fraumeni families, according to their constitutional TP53 mutation status. Families with germline missense mutations in the core DNA binding domain showed a more highly penetrant cancer phenotype than families with other TP53 mutations or no mutation. Cancer phenotype in families carrying such mutations was characterized by a higher cancer incidence and earlier ages at diagnosis, especially of breast cancer and brain tumours, compared with families carrying protein truncating or other inactivating mutations (P=0.03 for all cancers, P=0.006 for breast cancers, P=0.05 for brain tumours). Proband cancers showed significantly younger ages at diagnosis in those with missense mutations in the DNA binding domain than in those with protein inactivating mutations (P=0.031). In individuals with the former type of mutation, there was a significantly lower proportion of tumours which showed loss of the wild-type TP53 allele (P=0.004). These results are consistent with observations in experimental systems which demonstrate that certain mutations exhibit gain of function and/or dominant-negative properties. Our results support an enhanced oncogenic potential for such mutations in human populations.

Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

We report an extensive Li-Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT-PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.

Chromosome instability is a predominant trait of fibroblasts from Li-Fraumeni families.

Previous work has indicated a role for p53 in cell cycle control, genomic stability and cellular responses to DNA-damaging agents. However, few data are available for human fibroblasts heterozygous for defined germline mutations in TP53. We report studies on 25 strains derived from 12 families with Li-Fraumeni syndrome (LFS) and 18 strains from normal volunteers. The families include three that are classical LFS families, but in whom no TP53 mutation has been found. In the families with mutations, increased longevity and resistance to low-dose-rate ionizing radiation showed a statistically significant association with the presence of TP53 mutations. However, not all heterozygotes had increased longevity or were radioresistant, and fibroblasts from cancer-affected members of LFS families without TP53 mutations showed no significant increase in either of these end points. In contrast, all mutation-carrying strains showed evidence of genomic instability, expressed as aneuploidy, and accumulated structural chromosome aberrations in up to 100% of cells, usually accompanied by loss of the wild-type TP53 allele, immediately before senescence. Levels of aneuploidy higher than in normal cells were also observed in fibroblasts from families without TP53 mutations, suggesting that chromosome instability is a major factor in determining the cancer proneness of these families.

Germ-line mutations of TP53 in Li-Fraumeni families: an extended study of 39 families.

We have previously reported on the analysis of TP53 coding mutations in 12 classic Li-Fraumeni syndrome (LFS) families plus 9 families that were Li-Fraumeni-like (LFL) families (J. M. Birch et al., Cancer Res., 54: 1298-1304, 1994). Mutations were found in 6 of 12 LFS families and in 1 of 9 LFL families. We have now extended these studies to include an additional nine LFS and nine LFL families, and TP53 mutations have been detected in eight of nine LFS families and in three of nine LFL families. Six of the new mutations described here are the same as those previously identified in other Li-Fraumeni families and are missense mutations at codons 245, 248, and 273 (in two families); a nonsense mutation at codon 209; and a mutation at the splice donor site in exon 4. The other five mutations are novel germ-line mutations and include missense mutations at codons 136 and 344, a 2-bp deletion within codon 191, a splice acceptor mutation in intron 3, and a 167-bp deletion of part of exon 1 and intron 1. In addition, we have detected a codon 175 mutation in a family previously reported as TP53 negative. To summarize all of the data from the families we have studied in this and our previous report (J. M. Birch et al., Cancer Res., 54: 1298-1304, 1994), mutations have been detected in 15 of 21 LFS families (71%) and in 4 of 18 LFL families (22%). These figures are somewhat higher than those previously reported by us and others for the frequency of TP53 mutations in LFS and LFL families. This could reflect our analysis of all 11 exons of TP53, including noncoding regions, as well as the use of direct sequencing rather than other less-sensitive mutation detection methods.

An enzyme-linked immunosorbent assay using Schistosoma mansoni purified egg antigen for the diagnosis of schistosomiasis in Saudi Arabia.

The enzyme-linked immunosorbent assay (ELISA) using purified Schistosoma mansoni egg antigen (CEF6) was compared with standard parasitological diagnostic tests (potassium hydroxide digestion of faeces and urine filtration) for diagnosis of schistosomiasis in humans in Saudi Arabia. Faecal, urine and finger-prick blood samples were collected from 2 groups of individuals of both sexes, aged 1-50 years, in 2 areas in the western region of Saudi Arabia: 983 in a schistosomiasis endemic area (Al-Hijaz highlands) and 192 in a non-endemic area (Jeddah). In the non-endemic area, almost 90% of the blood samples gave optical density readings at 492 nm (OD) < 0.25. The mean OD in the schistosome endemic area (0.31) was much higher than in the non-endemic area (0.14). The prevalence of S. mansoni infection by faecal examination in the endemic area was 10.2% and the specificity and sensitivity of the ELISA using a cut-off OD of 0.25 were 55% and 90%, respectively. In the endemic area, there was a positive correlation between egg intensity and OD value. No S. haematobium was detected. In the non-endemic area, the specificity was 90%. The main reasons for false positive results may have been inapparent or cured S. mansoni infection.

A detailed study of loss of heterozygosity on chromosome 17 in tumours from Li-Fraumeni patients carrying a mutation to the TP53 gene.

We have studied a total of 36 tumours from 28 patients with germline mutations to the TP53 gene for loss of heterozygosity at TP53 using techniques of both direct sequencing and restriction fragment length polymorphism analysis. All patients were from families conforming to the definition of classical Li-Fraumeni syndrome (LFS) or were Li-Fraumeni-like (LFL). The data we have obtained show that loss of the wild-type TP53 gene is observed in under half (44%) of all tumours, and that the pattern of LOH at TP53 may be mutation specific. LOH has been observed in premalignant as well as invasive tumours. Two tumours (6%) show loss of the mutant allele and retention of the wild-type. To confirm that TP53 is indeed the target for LOH events on chromosome 17, we have used additional microsatellite repeats to examine patterns of allelic imbalance along the length of chromosome 17. Data from this analysis indicate that TP53 is the target of loss, but reveal some other interesting patterns of allelic imbalance at other loci on chromosome 17.

A novel deletion within exon 6 of TP53 in a family with Li-Fraumeni-like syndrome, and LOH in a benign lesion from a mutation carrier.

We report here a family with some of the characteristics of Li-Fraumeni syndrome (Li-Fraumeni-like) in which there is a 2 base pair deletion within exon 6 of TP53 in two affected individuals. Of particular interest in this family is a study of loss of heterozygosity (LOH) of the TP53 gene, and the finding that there is LOH in all cancers available for study from mutation carriers, and additionally from a benign endometrial polyp from one of those patients. Two other family members, one with a rectal carcinoma aged 55, the other with two separate benign lesions under the age of 45, were both wild-type for the TP53 mutation.

An extended Li-Fraumeni kindred with gastric carcinoma and a codon 175 mutation in TP53.

We present an extended family with Li-Fraumeni syndrome characterised by gastric and breast carcinoma, glioma, sarcoma, and leukaemia. This family showed strong evidence of linkage to TP53, and three of four tumours analysed showed loss of the wild type allele. A codon 175 missense mutation was identified in exon 5 in all available affected subjects. Counselling, screening, and issues surrounding presymptomatic testing are discussed.

Linkage studies in a Li-Fraumeni family with increased expression of p53 protein but no germline mutation in p53.

We report a family with the Li-Fraumeni syndrome (LFS) in whom we have been unable to detect a mutation in the coding sequence of the p53 gene. Analysis of linkage to three polymorphic markers within p53 enabled direct involvement of p53 to be excluded. This is the first example of a LFS family in whom exclusion of p53 has been possible. Four affected members of the family with sarcoma or premenopausal breast cancer showed increased expression of p53 protein in their normal tissues as detected by immunohistochemistry. It therefore appears that the LFS phenotype has been conferred by an aberrant gene, showing a dominant pattern of inheritance, which may be acting to compromise normal p53 function rather than by a mutation in p53 itself. In order to try to determine the chromosomal location of this putative gene, we have carried out studies of linkage to candidate loci. By these means we have excluded involvement of Rb1 and BRCA1 on chromosomes 13q and 17q respectively. The MDM2 oncogene on chromosome 12q was considered to be the prime candidate as MDM2 is amplified in sarcomas and the MDM2 product binds to p53. Furthermore, p53 mutation and amplification of MDM2 have been shown to be mutually exclusive events in tumour development. Linkage analysis to two polymorphic markers within MDM2 yielded a three-point LOD score of -5.4 at a recombination fraction theta equal to zero. Therefore MDM2 could be excluded. It is possible that the gene which is responsible for cancer susceptibility in this family, possibly via interaction with p53, will be important in the histogenesis of breast cancer in general. We are now carrying out further studies to locate and identify this gene.

Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families.

The entire coding sequence of the p53 gene was analysed for the presence of mutations in 12 families conforming to a restricted definition of Li-Fraumeni syndrome (classic LFS) and nine families with features of LFS conforming to a broader definition. Mutations were detected in seven families. Six were point mutations with one each affecting codons 175, 180, and 220 and three affecting codon 248. The seventh was a deletion/insertion mutation in exon 4. Germline mutations in p53 were a feature of families which included children with rhabdomyosarcoma and/or adrenal cortical carcinoma. Germline p53 mutations were detected in six of the nine families with such tumors. An analysis of these 7 mutations, together with 34 published examples, showed that more than one-half were transitions at CpG dinucleotides, suggesting that the majority of germline p53 mutations may arise as a result of spontaneous events. The most common cancers occurring in the 41 families with germline p53 mutations, in common with classic LFS, were bone and soft tissue sarcoma, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma, although less than one-half of the probands with germline p53 mutations came from classic LFS families. More than one-half of the cancers overall and nearly one-third of the breast cancers were diagnosed before 30 years of age. These observations have important implications for asymptomatic carriers of germline p53 mutations, and there is a need for international collaboration in the development of protocols for the management of such families.

Wilms' tumor in the Li-Fraumeni cancer family syndrome.

Scrutiny of the family pedigrees of a population-based series of 176 children diagnosed with Wilms' tumor between 1954 and 1990, along with a review of the literature on the Li-Fraumeni cancer family syndrome, indicate that Wilms' tumor may be an uncommon component of the syndrome and that a small proportion of children with Wilms' tumor may be members of Li-Fraumeni syndrome (LFS) families.