Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia.

PURPOSE: RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a phase I study of RG7112 in patients with hematologic malignancies was conducted. EXPERIMENTAL DESIGN: Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics; pharmacodynamics, such as TP53-mutation status and MDM2 expression; and preliminary clinical activity. Patients were divided into two cohorts: Stratum A [relapsed/refractory acute myeloid leukemia (AML; except acute promyelocytic leukemia), acute lymphoblastic leukemia, and chronic myelogenous leukemia] and Stratum B (relapsed/refractory chronic lymphocytic leukemia/small cell lymphocytic leukemia; CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity. RESULTS: RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 dose-limiting toxicities were reported. Pharmacokinetic analysis indicated that twice-daily dosing enhanced daily exposure. Antileukemia activity was observed in the 30 patients with AML assessed at the MTD, including 5 patients who met International Working Group (IWG) criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response. CONCLUSIONS: RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies.

Distress and psychosocial needs of a heterogeneous high risk familial cancer population.

In order to assess the levels of distress and psychosocial support needs of a high risk population, we undertook a study to look at both the objective and subjective levels of distress and the wants and needs of individuals from a high familial cancer risk population. Three hundred and eighteen individuals (160 affected, 158 unaffected) completed several distress and psychosocial needs questionnaires (including the Brief Symptom Inventory-18). Sixty key informants were also surveyed about their perspective on the support needs of this population. In the largely female (90%), largely HBOC syndrome group (approximately 90%), 20% had significant levels of generalized distress, with no significant differences between affected and unaffected individuals. Generalized distress was also not significantly different as a function of mutation status. Individuals who received inconclusive test results, however, were more likely to indicate somatic symptoms of distress. Those individuals who did not have social support were more likely to be those who had never had cancer and who either had a mutation, received inconclusive test results, or were not tested. Key informants were most likely to indicate that patients need more support. These results provide evidence for the importance of establishing regular psychosocial distress screening, including a focus on somatic symptoms, in such high risk populations.

Muenke syndrome with pigmentary disorder and probable hemimegalencephaly: An expansion of the phenotype.

We describe a 2-year-old boy born to healthy, consanguineous parents. He had craniofacial asymmetry with left frontal bossing, midface hypoplasia, proptosis, and low-set ears. In addition, he had curly, light hair, and oval hypomelanotic patches in the abdomen, lower limbs and back and one hyperpigmented patch in the groin without acanthosis nigricans. Cranial three-dimensional CT scan showed right-coronal, sagittal, and lambdoid suture synostoses. His cranial MRI at 2-months of age showed left hemimegalencephaly, hypoplasia of corpus callosum, and an abnormal configuration of hippocampus. In spite of these cranial findings, he had mild developmental delay and his neurological examination showed symmetric strength, tone and reflexes. Apart from febrile seizures, there was no history of epilepsy. The proband developed asymmetric hydrocephalus at the age of 18 months that required third ventriculostomy. Post-operative cranial MRI showed Chiari I- like malformation and asymmetry of cerebral hemispheres but less dysplastic cerebral cortex. Mutation analysis of FGFR3 showed a c.749C > G, p.Pro250Arg substitution. To the best of our knowledge, these manifestations have not been reported in patients with Muenke syndrome.

Polymorphisms in multiple genes are associated with resting heart rate in a stepwise allele-dependent manner.

OBJECTIVE: The purpose of this study was to use a candidate gene approach to identify common polymorphisms that are associated with resting sinus heart rate in a population without overt cardiovascular disease. BACKGROUND: Increased resting heart rate is significantly associated with susceptibility to development of myocardial infarction, sudden cardiac death, and overall cardiac mortality. METHODS: A longitudinal cohort of 1468 individuals (active and retired middle-aged Canadian firefighters) who were enrolled in the Firefighters and Their Endothelium (FATE) study was evaluated. Resting heart rate was recorded from the electrocardiogram (ECG) obtained at enrollment. Candidate genes were selected for their known roles in sinus node automaticity and/or its regulation, and single nucleotide polymorphisms (SNPs) with a minor allele frequency of > or =0.20 were targeted. A total of 53 SNPs in 46 genes were selected and analyzed in a screening sample, and 33 SNPs in 29 genes were evaluated in the full population. RESULTS: Univariate analysis detected five putative associations between HR and SNPs. As expected, environmental covariates were identified. Three polymorphisms, ADRB1 G389R, SCN5a H558R, and CASQ1 intron 2, remained statistically significant and independent of covariates. Some alleles were associated with higher and some with lower heart rates. A stepwise increase in heart rate was observed that was dependent on the number of tachycardia-associated alleles with progressive increases in mean heart rate from 51 to 66 bpm. CONCLUSIONS: Common polymorphisms are associated with heart rate in a stepwise allele-dependent manner.

Evaluation of BRCA1 and BRCA2 mutation prevalence, risk prediction models and a multistep testing approach in French-Canadian families with high risk of breast and ovarian cancer.

BACKGROUND AND OBJECTIVE: In clinical settings with fixed resources allocated to predictive genetic testing for high-risk cancer predisposition genes, optimal strategies for mutation screening programmes are critically important. These depend on the mutation spectrum found in the population under consideration and the frequency of mutations detected as a function of the personal and family history of cancer, which are both affected by the presence of founder mutations and demographic characteristics of the underlying population. The results of multistep genetic testing for mutations in BRCA1 or BRCA2 in a large series of families with breast cancer in the French-Canadian population of Quebec, Canada are reported. METHODS: A total of 256 high-risk families were ascertained from regional familial cancer clinics throughout the province of Quebec. Initially, families were tested for a panel of specific mutations known to occur in this population. Families in which no mutation was identified were then comprehensively tested. Three algorithms to predict the presence of mutations were evaluated, including the prevalence tables provided by Myriad Genetics Laboratories, the Manchester Scoring System and a logistic regression approach based on the data from this study. RESULTS: 8 of the 15 distinct mutations found in 62 BRCA1/BRCA2-positive families had never been previously reported in this population, whereas 82% carried 1 of the 4 mutations currently observed in > or =2 families. In the subset of 191 families in which at least 1 affected individual was tested, 29% carried a mutation. Of these 27 BRCA1-positive and 29 BRCA2-positive families, 48 (86%) were found to harbour a mutation detected by the initial test. Among the remaining 143 inconclusive families, all 8 families found to have a mutation after complete sequencing had Manchester Scores > or =18. The logistic regression and Manchester Scores provided equal predictive power, and both were significantly better than the Myriad Genetics Laboratories prevalence tables (p<0.001). A threshold of Manchester Score > or =18 provided an overall sensitivity of 86% and a specificity of 82%, with a positive predictive value of 66% in this population. CONCLUSION: In this population, a testing strategy with an initial test using a panel of reported recurrent mutations, followed by full sequencing in families with Manchester Scores > or =18, represents an efficient test in terms of overall cost and sensitivity.

Comments on 'Flow dependence and repeatability of interrupter resistance in lower airways and nose'.

The authors (Eiser et al 2005 Physiol. Meas. 26 143-56) have undertaken a much needed investigation into the extent of the flow dependence of resistance, as measured by the interrupter technique (Rint). The clear explanations of the general principles should prove invaluable to the readers. However, I should like to highlight some small technical errors as well as raising some points of concern regarding the accuracy of the findings due to the relatively slow sampling rate for flow and subjects being asked to breathe 'deeper and faster', if the device was not triggering at higher flows.

The effect of iron status on vascular health.

The effect of increased iron stores on the progression of atherosclerosis and endothelial health remains inconclusive. This study was designed to evaluate the relationship between hemochromatosis genotypes, serum ferritin levels and presymptomatic vascular abnormalities in a cohort of healthy subjects. Carotid intima-media thickness (CIMT) and brachial flow-mediated vasodilation (FMD) were assessed by high-resolution ultrasound in 907 male (47 +/- 10 years) participants enrolled in the Firefighters and their Endothelium (FATE) study. Analyses of the hemochromatosis C282Y, H63D and S65C alleles were simultaneously determined by a single nucleotide polymorphism (SNP) primer extension method. It was found that brachial FMD was not related to serum ferritin or hemochromatosis genotype status. The presence of a hemochromatosis-associated genotype (n = 18) or heterozygosity for the C282Y genotype (n = 98) was not associated with an increased mean CIMT. After adjustment for conventional risk factors, serum ferritin was also not associated with mean CIMT. In conclusion, neither ferritin nor a hemochromatosis genotype was related to brachial endothelial function or carotid atherosclerosis. The present study does not support the hypothesis that mild to moderately increased iron stores are associated with enhanced atherosclerosis risk.

No Evidence of BRCA1/2 genomic rearrangements in high-risk French-Canadian breast/ovarian cancer families.

The discovery of deleterious mutations in the breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, has facilitated the identification of individuals at particularly high risk of these diseases. There is a wide variation between populations in the prevalence and related risks of various types of BRCA1/2 mutations, so estimates cannot be extrapolated to Canadians, especially not founder populations such as French- Canadians. Polymerase chain reaction (PCR)-based methods were used to detect the majority of these mutations. These approaches usually failed to detect large DNA rearrangements, which have been claimed to be involved in other populations in 5% to up to 36% of BRCA1-positive families. There is very little information about the contribution of this type of mutation in BRCA2-positive families. To investigate if our available mutation spectrum of BRCA1 and BRCA2 in high-risk French-Canadian breast/ovarian cancer families has been biased by PCR-based direct sequencing methods, we first used Southern blot analysis to test DNA samples from 61 affected/obligate carrier individuals from 58 families in which no BRCA1/2 deleterious mutation was found. Finally, 154 individuals from 135 BRCA1/2 nonconclusive families, including all those tested previously by Southern blot analysis, were tested with the new multiplex ligation probe amplification (MLPA) technique. These approaches failed to detect any rearrangement. Moreover, if the frequency of MLPA-detectable rearrangements in our cohort of 135 BRCA1/2 nonconclusive families was 2.2% or higher, we would have had a 95% or greater chance of observing at least one such rearrangement. As no rearrangements were identified, such large rearrangements must be quite rare in our population.

Partnering in oncogenetic research--the INHERIT BRCAs experience: opportunities and challenges.

Today it is common to conduct research in collaboration with colleagues from different disciplines and institutions. The INterdisciplinary HEalth Research International Team on BReast CAncer susceptibility (INHERIT BRCAs), involves Canadian and international experts from diverse fields working with health service providers, patients and collaborators from the World Health Organization and other European networks. Evidence-based information and knowledge transfer drive our efforts to advance genomic research to understand the genetic basis of cancer susceptibility and treatment response. Several goals reveal the interdisciplinary team approach: (a) to estimate the prevalence and penetrance of BRCA1 and BRCA2 mutations and their deleterious impact upon different populations; (b) to pinpoint novel breast cancer susceptibility loci; (c) to assess the efficacy of clinical interventions; (d) to address changes in quality of life and health-related behaviour from the decision to undergo genetics testing and during follow-up; (e) to evaluate legal, social and ethical implications; and, finally; (f) to promote professional and public education by facilitating the transfer of research findings to clinical practice and informing policy makers. The lessons learned by the INHERIT research team and future challenges are presented.

No evidence of false reassurance among women with an inconclusive BRCA1/2 genetic test result.

BACKGROUND: Little is known about how women who receive an inconclusive result from BRCA1/2 testing interpret their result. Clinical observations suggest that some of them may be falsely reassured and, consequently, may not adhere to recommended surveillance. The purpose of this study is to evaluate whether women with inconclusive BRCA1/2 test results are falsely reassured. METHODS: Participants were adult women with a family history suggestive of a germ-line mutation in either the BRCA1 or the BRCA2 gene who underwent genetic testing in the context of the interdisciplinary research program INHERIT BRCAs. Data were collected using self-administered questionnaires at genetic counseling and 1 month after result disclosure. Reassurance was assessed through indicators of cancer risk perception, cancer worry, relief following result disclosure, painfulness of the test result, and its effect on quality of life. RESULTS: Five-hundred women (105 carriers, 140 noncarriers, and 255 inconclusive) were included in this analysis. Compared to noncarriers, women with inconclusive results had higher cancer risk perception, were more worried about cancer, were less relieved by their test result, and perceived their quality of life as being more adversely affected by it. CONCLUSION: The differences observed between noncarriers and women who received an inconclusive result run counter to the hypothesis that the latter are falsely reassured following BRCA1/2 testing. For clinicians, our findings show the value of taking precautions to fully explain to women that inconclusive results do not rule out the possibility that they still may face a higher risk of developing breast and/or ovarian cancer.

A new alternative splice variant of BRCA1 containing an additional in-frame exon.

The breast/ovarian cancer susceptibility gene BRCA1 interact with multiple protein complexes involved in cellular mechanisms, such as DNA repair, transcription, homologous recombination and cell cycle regulation. Extensive analyses over the past decade led to the detection of several BRCA1 alternative splice variants. Here, we identify the first BRCA1 alternative splice variant containing an additional in-frame exon. This previously unknown exon 13A-containing transcript is generated by the insertion of 66 nucleotides between exons 13 and 14, due to alternative splicing in intron 13 (IVS13-2786-2720). Furthermore, exon 13A-containing transcript was detectable in total RNA samples from 12 normal tissues and several breast and other cancer cell lines. As revealed by real-time PCR analysis, this transcript corresponds to 20 to 25% of the total BRCA1 mRNA expression levels in leukocytes, brain and cerebellum tissues, whereas its relative level of expression is less than 5% in other tested tissues and cancer cell lines. This novel alternative transcript adds 22 amino acids after residue 1452, thus modifying the primary structure of the trans-activation domain 1 (AD1) and the protein-protein interacting domain of BRCA1 with BRCA2, AR and MSH2. No sequence variant has been detected by direct genomic sequencing of exon 13A in individuals originating from high-risk breast/ovarian cancer families.

Pressure oscillation amplitude after interruption of tidal breathing as an index of change in airway mechanics in preschool children.

Bronchodilator reversibility testing using change in airway resistance during interruption (Rint) is feasible in preschool children. Analysis of postocclusion oscillations of the mouth pressure-time transient (Pmo(t)), recorded during airflow interruption, may offer an alternative index of change in airway mechanics. We analyzed Pmo(t) oscillation amplitude in three different ways: 1) difference between the first relative maximum and minimum (AMxMn); 2) detection of the dominant frequency using Fourier analysis (AFS); and 3) curve-fitting based on a mathematical model (ACurv). In 25 asymptomatic asthmatic children, aged 2.5-5.6 years, who had undertaken reversibility testing, the correlation coefficients between baseline Rint and amplitude were: AMxMn r = -0.84, AFS r = -0.82, ACurv r = -0.84. The coefficient of variation (CoV) of readings contributing to baseline Rint measurement, as median (range), was 12% (5-24%), which was not significantly different from AFS or ACurv (P > 0.05). All parameters were significantly different postbronchodilator (P < 0.001). Using the sensitivity index, i.e., the change after intervention divided by the baseline standard deviation, ACurv was the most sensitive and Rint the least sensitive, with median (range) at 2.72 (-0.84 to 12.10) and 1.91 (-1.17 to 9.50), respectively (P = 0.005). Our results suggest that oscillation amplitude analysis may provide a sensitive index of change in airway mechanics in preschool children undertaking bronchodilator reversibility testing.

Molecular and genealogical characterization of the R1443X BRCA1 mutation in high-risk French-Canadian breast/ovarian cancer families.

The Quebec population contains about six-million French Canadians, descended from the French settlers who colonized "Nouvelle-France" between 1608 and 1765. Although the relative genetic contribution of each of these founders is highly variable, altogether they account for the major part of the contemporary French-Canadian gene pool. This study was designed to analyze the role of this founder effect in the introduction and diffusion of the BRCA1 recurrent R1443X mutant allele. A highly conserved haplotype, observed in 18 French-Canadian families and generated using 17 microsatellite markers surrounding the BRCA1 locus, supports the fact that the R1443X mutation is a founder mutation in the Quebec population. We also performed haplotyping analysis of R1443X carriers on 19 other families from seven different nationalities; although the same alleles are shared for three markers surrounding the BRCA1 gene, distinct haplotypes were obtained in four families, suggesting multiple origins for the R1443X mutation. Ascending genealogies of the 18 French Canadian families and of controls were reconstructed on an average depth of 10 generations. We identified the founder couple with the highest probability of having introduced the mutation in the population. Based on the descending genealogy of this couple, we detected the presence of geographical concentration in the diffusion pattern of the mutation. This study demonstrates how molecular genetics and demogenetic analyses can complement each other to provide findings that could have an impact on public health. Moreover, this approach is certainly not unique to breast cancer genetics and could be used to understand other complex traits.

Adult onset spinocerebellar ataxia in a Canadian movement disorders clinic.

BACKGROUND: The spinocerebellar ataxias (SCAs) are a genetically and clinically heterogeneous group of neurodegenerative disorders. Relative frequencies vary within different ethnic groups and geographical locations. OBJECTIVES: 1) To determine the frequencies of hereditary and sporadic adult onset SCAs in the Movement Disorders population; 2) to assess if the fragile X mental retardation gene 1 (FMR1) premutation is found in this population. METHODS: A retrospective chart review of individuals with a diagnosis of adult onset SCA was carried out. Testing for SCA types 1, 2, 3, 6, 7, and 8, Dentatorubral-pallidoluysian atrophy (DRPLA), Friedreich ataxia and the FMR1 expansion was performed. RESULTS: A total of 69 patients in 60 families were identified. Twenty-one (35%) of the families displayed autosomal dominant and two (3.3%) showed autosomal recessive (AR) pattern of inheritance. A positive but undefined family history was noted in nine (15%). The disorder appeared sporadic in 26 patients (43.3%). In the AD families, the most common mutation was SCA3 (23.8%) followed by SCA2 (14.3%) and SCA6 (14.3%). The SCA1 and SCA8 were each identified in 4.8%. FA was found in a pseudodominant pedigree, and one autosomal recessive pedigree. One sporadic patient had a positive test (SCA3).Dentatorubral-pallidoluysian atrophy and FMR1 testing was negative. CONCLUSION: A positive family history was present in 53.3% of our adult onset SCA patients. A specific genetic diagnosis could be given in 61.9% of dominant pedigrees with SCA3 being the most common mutation, followed by SCA2 and SCA6. The yield in sporadic cases was low. The fragile X premutation was not found to be responsible for SCA.

Cortical gray matter volumes are associated with subjective responses to cocaine infusion.

We analyzed the relationship between cocaine-induced euphoria and measures of frontal and temporal gray matter volumes in eleven cocaine-dependent (CD) patients who underwent magnetic resonance imaging (MRI). Self-reported ratings of the intensity of euphoric response to cocaine infusion were obtained from the CD subjects at 3, 10, and 30 minutes after cocaine infusion. Significant positive correlation between frontal and temporal cortical gray matter volume and the intensity of euphoria was observed at 10 minutes after IV cocaine. The data suggest that frontal and temporal lobe gray matter volume is associated with some of the reinforcing effects of cocaine. Given the well-established negative linear relationship between cortical gray matter volume and age, cortical gray matter volume may be a marker for the neurobiological substrate of the age-related reduction in addiction rates.

Vitamin B12 decreases, but does not normalize, homocysteine and methylmalonic acid in end-stage renal disease: a link with glycine metabolism and possible explanation of hyperhomocysteinemia in end-stage renal disease.

The genetic and environmental factors influencing catabolism of homocysteine in end-stage renal disease (ESRD) patients remain poorly understood. This study investigated how genetic and nutritional influences affect the response to high-dose vitamin B(12) and folate treatment in ESRD patients with hyperhomocysteinemia. We studied 81 hemodialysis patients with hyperhomocysteinemia (> 16 micromol/L) on varied doses of a multivitamin containing 1 mg of folic acid per day. After screening blood work, all patients were switched to daily multivitamin therapy including 1 mg of folic acid for 4 weeks. Vitamin B(12), 1 mg/d, was added for an additional 4 weeks. Patients were then randomized to receive folic acid or placebo. The influence of the 3 methylenetetrahydrofolate reductase (MTHFR) 677 C-->T genotypes on the efficacy of vitamin therapy was assessed. In addition, we investigated how the metabolic complications of ESRD, including the relationship between methylmalonic acid (MMA) and circulating glycine, may contribute to hyperhomocysteinemia. There was no significant difference in total homocysteine (tHcy) levels between the MTHFR 677 C-->T genotypes during the screening phase of the trial. Treatment with a daily multivitamin containing 1 mg folate significantly lowered tHcy levels in all patients by 19.2%. Further supplementation with 1 mg vitamin B(12) resulted in greater tHcy reduction among subjects with the MTHFR 677 T/T genotype (P<.01, T/T v C/C or C/T) while lowering MMA equally in all MTHFR genotypes. There was a significant positive correlation between plasma glycine levels and MMA (P <.05). High-dose vitamin therapy significantly lowers, but does not normalize, MMA and tHcy levels. The MTHFR genotype, while influencing homocysteine levels, was not responsible for the majority of the elevation in plasma tHcy.

The fate of endothelial function testing: rationale and design of the Firefighters And Their Endothelium (FATE) study.

Endothelial dysfunction plays a pivotal role in the development and progression of atherosclerotic vascular disease. The endothelium is strategically located between blood and vascular smooth muscle, making it both vulnerable to a variety of injurious stimuli but also available for interrogation as a marker of vascular health. Firefighters And Their Endothelium (FATE) is a prospective, longitudinal cohort study designed to assess the relationship between endothelial function, emerging cardiovascular risk factors and ultimately atherosclerotic vascular disease. It is hypothesized that participants with impaired endothelial function will be at increased risk of atherosclerotic complications. This Canadian initiative will recruit 1600 middle-aged participants with no known history of cardiovascular disease to be followed for cardiovascular events over the next decade. Quantitative B-mode ultrasound will be employed to assess endothelial function and subclinical atherosclerosis. This research is designed to redefine the approach to the primary prevention of atherosclerosis.