Magnetic resonance imaging (MRI) of pharmacological ascorbate-induced iron redox state as a biomarker in subjects undergoing radio-chemotherapy.

Pharmacological ascorbate (P-AscH-) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe3+) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe2+). Because paramagnetic Fe3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH--induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe3+ or Fe2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH- plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH- infusion, post-P-AscH- infusion, and post-radiation (3-4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe3+ and Fe2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe2+ phantoms. Compared to pre-P-AscH-, GBM T2* and QSM imaging were significantly changed post-P-AscH- infusion consistent with conversion of Fe3+ to Fe2+. No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH- inducing greater changes in tumor labile iron pools compared to the normal tissue. CONCLUSIONS: T2* and QSM MR-imaging responses are consistent with P-AscH- reducing Fe3+ to Fe2+, selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH--induced changes in redox-active iron.

First-in-Human Phase I Clinical Trial of Pharmacologic Ascorbate Combined with Radiation and Temozolomide for Newly Diagnosed Glioblastoma.

PURPOSE: Standard treatment for glioblastoma (GBM) includes surgery, radiation therapy (RT), and temozolomide (TMZ), yielding a median overall survival (OS) of approximately 14 months. Preclinical models suggest that pharmacologic ascorbate (P-AscH-) enhances RT/TMZ antitumor effect in GBM. We evaluated the safety of adding P-AscH- to standard RT/TMZ therapy. PATIENTS AND METHODS: This first-in-human trial was divided into an RT phase (concurrent RT/TMZ/P-AscH-) and an adjuvant (ADJ) phase (post RT/TMZ/P-AscH- phase). Eight P-AscH- dose cohorts were evaluated in the RT phase until targeted plasma ascorbate levels were achieved (≥20 mmol/L). In the ADJ phase, P-AscH- doses were escalated in each subject at each cycle until plasma concentrations were ≥20 mmol/L. P-AscH- was infused 3 times weekly during the RT phase and 2 times weekly during the ADJ phase continuing for six cycles or until disease progression. Adverse events were quantified by CTCAE (v4.03). RESULTS: Eleven subjects were evaluable. No dose-limiting toxicities occurred. Observed toxicities were consistent with historical controls. Adverse events related to study drug were dry mouth and chills. Targeted ascorbate plasma levels of 20 mmol/L were achieved in the 87.5 g cohort; diminishing returns were realized in higher dose cohorts. Median progression-free survival (PFS) was 9.4 months and median OS was 18 months. In subjects with undetectable MGMT promoter methylation (n = 8), median PFS was 10 months and median OS was 23 months. CONCLUSIONS: P-AscH-/RT/TMZ is safe with promising clinical outcomes warranting further investigation.

Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate.

Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in >≈10 mM plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 lM. However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 mM. The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (mM) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At -20°C, plasma concentrations are relatively stable for 2-3 weeks, while at -80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at -20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.

Reversion of AHRR Demethylation Is a Quantitative Biomarker of Smoking Cessation.

Smoking is the largest preventable cause of morbidity and mortality in the world. Although there are effective pharmacologic and behavioral treatments for smoking cessation, our inability to objectively quantify smokers' progress in decreasing smoking has been a barrier to both clinical and research efforts. In prior work, we and others have shown that DNA methylation at cg05575921, a CpG residue in the aryl hydrocarbon receptor repressor (AHRR), can be used to determine smoking status and infer cigarette consumption history. In this study, we serially assessed self-report and existing objective markers of cigarette consumption in 35 subjects undergoing smoking cessation therapy, then quantified DNA methylation at cg05575921 at study entry and three subsequent time points. Five subjects who reported serum cotinine and exhaled carbon monoxide verified smoking abstinence for the 3 months prior to study exit averaged a 5.9% increase in DNA methylation at cg05575921 (p < 0.004) over the 6-month study. Although the other 30 subjects did not achieve smoking cessation at the 6-month time point, their self-reported reduction of cigarette consumption (mean = 6 cigarettes/day) was associated with a 2.8% increase DNA methylation at cg05575921 (p < 0.05). Finally, a survey of subjects as they exited the study demonstrated strong support for the clinical use of epigenetic biomarkers. We conclude that AHRR methylation status is a quantifiable biomarker for progress in smoking cessation that could have substantial impact on both smoking cessation treatment and research.

A quantitative epigenetic approach for the assessment of cigarette consumption.

Smoking is the largest preventable cause of morbidity and mortality in the world. Despite the development of numerous preventive and treatment interventions, the rate of daily smoking in the United States is still approximately 22%. Effective psychosocial interventions and pharmacologic agents exist for the prevention and treatment of smoking. Unfortunately, both approaches are hindered by our inability to accurately quantify amount of cigarette consumption from the point of initial experimentation to the point of total dependency. Recently, we and others have demonstrated that smoking is associated with genome-wide changes in DNA methylation. However, whether this advance in basic science can be employed as a reliable assay that is useful for clinical diagnosis and treatment has not been shown. In this communication, we determine the sensitivity and specificity of five of the most consistently replicated CpG loci with respect to smoking status using data from a publically available dataset. We show that methylation status at a CpG locus in the aryl hydrocarbon receptor repressor, cg05575921, is both sensitive and specific for smoking status in adults with a receiver operated curve characteristic area under the curve of 0.99. Given recent demonstrations that methylation at this locus reflects both intensity of smoking and the degree of smoking cessation, we conclude that a methylation-based diagnostic at this locus could have a prominent role in understanding the impact of new products, such as e-cigarettes on initiation of cigarette smoking among adolescents, while improving the prevention and treatment of smoking, and smoking related disorders.

A pilot examination of the genome-wide DNA methylation signatures of subjects entering and exiting short-term alcohol dependence treatment programs.

Alcoholism has a profound impact on millions of people throughout the world. However, the ability to determine if a patient needs treatment is hindered by reliance on self-reporting and the clinician's capability to monitor the patient's response to treatment is challenged by the lack of reliable biomarkers. Using a genome-wide approach, we have previously shown that chronic alcohol use is associated with methylation changes in DNA from human cell lines. In this pilot study, we now examine DNA methylation in peripheral mononuclear cell DNA gathered from subjects as they enter and leave short-term alcohol treatment. When compared with abstinent controls, subjects with heavy alcohol use show widespread changes in DNA methylation that have a tendency to reverse with abstinence. Pathway analysis demonstrates that these changes map to gene networks involved in apoptosis. There is no significant overlap of the alcohol signature with the methylation signature previously derived for smoking. We conclude that DNA methylation may have future clinical utility in assessing acute alcohol use status and monitoring treatment response.

The relationship of deiodinase 1 genotype and thyroid function to lifetime history of major depression in three independent populations.

Major depression (MD) is often associated with disturbances of the hypothalamic/pituitary/thyroid (HPT) axis. Unfortunately, whether this association is secondary to common underlying genetic variation or whether the MD-associated disturbances in HPT function are chronic or state-dependent is unknown. To examine these questions, we genotyped 12 single nucleotide polymorphisms identified in previous genome wide association analyses of thyroid function in DNA contributed by 1,555 subjects from three longitudinal ethnically diverse studies that are well-characterized for lifetime MD and thyroid function. We then examined associations between genetic variants and key outcomes of thyroid stimulating hormone, free thyroxine (FT4) and depression. We confirmed prior findings that two variants in deiodinase 1 (DIO1), including a variant in the 3'UTR of DIO1 (rs11206244), were associated with altered FT4 levels in both White and African American subjects. We also found that rs11206244 genotype was associated with lifetime MD in White female subjects, in particular those from high-risk cohorts. However, we found no association of current FT4 levels with lifetime MD in either ethnic group. We conclude that genetic variation influencing thyroid function is a risk factor for MD. Given the evidence from prior studies, further investigations of role of HPT variation in etiology and treatment of MD are indicated.

Medical and psychiatric problems among men and women in a community corrections residential setting.

Though the medical and mental health morbidity of incarcerated offenders has been discussed in a number of recent reports, very few data have been published concerning medical and mental health problems facing those on community corrections supervision. In this study of community corrections offenders utilizing residential facilities, we found that frequencies of substance use disorders, other mental health disorders, and medical problems exceeded frequencies found in the community and, in some cases, were higher than frequencies found in incarcerated individuals. Of particular concern were the high frequencies of substance use disorders, traumatic brain injury, anxiety states, suicidal ideation, and prior self-harm. While the level of self-reported medical and mental health service utilization was higher than expected, it appeared low relative to the disease burden reported by this special population. We conclude that concurrent evaluation and treatment of medical and psychiatric problems during the process of community supervision is indicated in this population.

Role of GABRA2 on risk for alcohol, nicotine, and cannabis dependence in the Iowa Adoption Studies.

AIM: A number of studies have shown that genetic variation at GABRA2 alters vulnerability to alcohol dependence. The exact identity of the causal variant(s), and the relationship of these variants to other forms of substance use and behavioral illness is, however, uncertain. OBJECTIVE: Therefore, we genotyped 516 individuals from the Iowa Adoption Studies, a large longitudinal case and control adoption study of substance use, at 39 single nucleotide polymorphisms encompassing the GABRA2 locus and analyzed them with respect to their lifetime history of three common forms of substance use dependence [alcohol dependence (AD), nicotine dependence (ND), and cannabis dependence (CD)] in the Iowa Adoption Studies and relevant exposure variables. RESULT: Using regression analysis, we found substantial evidence that both GABRA2 genotype and haplotype are significantly related to vulnerability to AD, ND, and CD, with the strongest relationships noted with respect to ND. Consistent with earlier studies suggesting exposure is an important step in the development of substance use, we found the inclusion of substance exposure data in our analytic models markedly increased the strength of the genetic associations of GABRA2 haplotype with substance use. Finally, we report that the genetic effects were markedly more pronounced in females than in males. CONCLUSION: We conclude that genetic variation at or near the GABRA2 locus significantly affects vulnerability not only to AD, but to other forms of substance use including ND and CD, and that the effects may be sex dependent.

Examination of the nicotine dependence (NICSNP) consortium findings in the Iowa adoption studies population.

INTRODUCTION: Nicotine dependence results from a complex interplay of genetic and environmental factors. Over the past several years, a large number of studies have been performed to identify distinct gene loci containing genetic vulnerability to nicotine dependence. Two of the most prominent studies were conducted by the Collaborative Study of the Genetics of Nicotine Dependence (NICSNP) Consortium using both candidate gene and high-density association approaches. METHODS: We attempted to confirm and extend the most significant findings from the high-density association study and the candidate gene study using the behavioral and genetic resources of the Iowa Adoption Studies, the largest case-control adoption study of substance use in the United States. RESULTS: We found evidence that genetic variation at CHRNA1, CHRNA2, CHRNA7, and CHRNB1 alters susceptibility to nicotine dependence, but we did not replicate any of the most significant single nucleotide polymorphism associations from the NICSNP high-density association study. DISCUSSION: Further examination of the NICSNP findings in other population samples is indicated.

The relationship of 5HTT (SLC6A4) methylation and genotype on mRNA expression and liability to major depression and alcohol dependence in subjects from the Iowa Adoption Studies.

Serotonin Transporter (5HTT or SLC6A4) mRNA transcription is regulated by both genetic and epigenetic mechanisms. Unfortunately, despite intense scrutiny, the exact identity and contribution of each of these regulatory mechanisms, and their relationship to behavioral illness remain unknown. This lack of knowledge is critical because alterations in SLC6A4 function are posited to be central to a wide variety of CNS disorders. In order to address this shortcoming, we quantified 5HTTLPR genotype, SLC6A4 mRNA production and CpG methylation using biomaterial from 192 lymphoblast cell lines derived from subjects who participated in the latest wave of the Iowa Adoption Studies. We then analyzed the resulting data with respect to clinical characteristics. We confirmed prior findings that the short (s) 5HTTLPR allele is associated with lower amounts of mRNA transcription, but there was no significant effect of the "Long G" allele on mRNA transcription. We also found that CpG methylation was higher (P < 0.0008) and mRNA production (P < 0.0001) was lower in females as compared to males. Those subjects with a lifetime history of Alcohol Dependence had higher levels of SLC6A4 mRNA. There was a trend for an association of increased overall methylation with lifetime history of major depression. Finally, we confirm our prior findings that the exact levels of 5HTT mRNA expression are dependent on how it is measured. We conclude that both genetic variation and epigenetic modifications contribute to the regulation of SLC6A4 function and that more in-depth studies of the molecular mechanisms controlling gene activity and the relationship of these mechanisms to behavioral illness are indicated.

Transcriptional profiling of subjects from the Iowa adoption studies.

Transcriptional profiling has been used to identify gene expression patterns indicative of general medical illnesses such as atherosclerosis. However, whether these methods can identify common psychiatric disorders has not been established. To answer this question with respect to nicotine use, we used genome-wide expression profiling lymphoblast cell lines from six actively smoking Iowa Adoption Studies (IAS) subjects and nine "clean" control subjects, followed by real-time PCR (RT-PCR) of gene expression patterns in lymphoblast derived RNA from 94 subjects in the IAS. As compared to those from controls without a history of smoking (n = 9), the expression levels of 579 of 29,098 genes were significantly up-regulated and expression levels of 584 of 29,098 genes were significantly down-regulated in lymphoblast lines from currently smoking subjects (n = 6). RT-PCR confirmation of four select RNA levels confirmed the validity of the overall profile and revealed highly significant relationships between the expression of some of these transcripts and (1) major depression, (2) antisocial personality, (3) nicotine dependence, and (4) cannabis dependence. We conclude that the use of expression profiling may contribute significant insights into the biology of complex behavioral disorders.

No association of the C677T methylenetetrahydrofolate reductase polymorphism with schizophrenia.

Some serological and genetic studies have suggested that alterations in folate metabolism are associated with increased vulnerability to schizophrenia. In particular, these findings are most striking for the role of a putatively functional variant (C677T) in the methylenetetrahydrofolate reductase (MTHFR) gene. To test the hypothesis that the T allele and the TT genotype are risk factors for psychosis, we genotyped the C677T polymorphism in 206 participants with schizophrenia or schizoaffective disorder and 359 participants from a population control sample. Neither the T allele nor the TT genotype was associated with increased risk for schizophrenia. These results do not support a role for the C677T MTHFR variant in schizophrenia.

An association study of PCQAP polymorphisms and schizophrenia.

INTRODUCTION: PCQAP is a member of the mediator family of transcription co-activators that is found in the region of 22q11, which is consistently deleted in DiGeorges/velocranialfacial (VCF) syndrome. As such, it is a gene of interest to behavioral geneticists because VCF is also associated with a high rate of psychosis and because defects in other mediator genes have been linked to psychosis and abnormal neurodevelopmental abnormalities. Recently, DeLuca and colleagues reported that polymorphisms in a trinucleotide repeat in exon 7 of PCQAP were associated with schizophrenia in a case-control study of Italian schizophrenics. OBJECTIVE AND METHODS: To confirm and extend the prior findings, we conducted a case-control association analysis using DNA from 233 schizophrenics and 371 random controls. RESULTS: Unfortunately, we did not find any significant differences in the distribution of CAG repeat alleles between subjects and controls. CONCLUSIONS: These findings limit the role of exon 7 PCQAP polymorphisms in the pathogenesis of schizophrenia.