Lesley J. Ashton

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN amplified neuroblastoma

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′ untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.

ABCC Multidrug Transporters in Childhood Neuroblastoma: Clinical and Biological Effects Independent of Cytotoxic Drug Efflux

Background Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. Methods A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)–driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN1/−, 205 hMYCN+/1 mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan–Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. Results Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the “poor prognosis” expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). Conclusion ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

Buccal DNA Collection: Comparison of Buccal Swabs with FTA Cards

Collection and analysis of DNA, most commonly from blood or buccal cells, is becoming more common in epidemiologic studies. Buccal samples, which are painless to take and relatively easily collected, are often the preferred source. There are several buccal cell collection methods: swabs, brushes, mouthwash, and treated cards, such as FTA or IsoCode cards. Few studies have systematically compared methods of buccal cell collection with respect to DNA yield and amplification success under similar conditions. We compared buccal DNA collection and amplification using buccal swabs and FTA cards in 122 control subjects from our Australian case-control study of childhood acute lymphoblastic leukaemia. Buccal DNA was quantified using a real-time PCR for β-actin and genotyped at the loci of three polymorphisms (MTHFR 677C>T, ACE I/D, and XPD 1012G>A). PCR was successful with DNA from buccal swabs for 62% to 89% of subjects and from FTA cards for 83% to 100% of subjects, depending on the locus. The matched pair odds ratios (95% confidence interval) comparing success of FTA cards with buccal swabs are as follows: MTHFR 677C>T using PCR-RFLP, 12.5 (11.6-13.5) and using real-time PCR, 130.0 (113.1-152.8); ACE I/D using PCR-amplified fragment length polymorphism, 3.36 (3.2-3.5); XPD 1012G>A using real-time PCR, 150.0 (132.7-172.3). FTA cards are a robust DNA collection method and generally produce DNA suitable for PCR more reliably than buccal swabs. There are, however, technical challenges in handling discs punched from FTA cards that intending users should be aware of. (Cancer Epidemiol Biomarkers Prev 2006;15(4):816–9)

Maternal use of Folic Acid and Other Supplements and Risk of Childhood Brain Tumors

Background: Interest in a possible protective effect of maternal vitamin use before or during pregnancy against childhood brain tumors (CBT) and other childhood cancers has grown over the past decade. Our Australian study of CBTs, conducted between 2005 and 2011, investigated whether maternal use folic acid and other supplements was protective. Methods: Case children were identified through the 10 Australian pediatric oncology centers and controls were recruited by national random digit dialing. Mothers of 327 cases and 867 control children provided information on supplement use before and during the index pregnancy, including brand name, dose, and timing. Data were analyzed using multivariable unconditional logistic regression. Results: The OR for any maternal use of folic acid, use of folic acid without iron or vitamins B6, B12, C, or A, and any vitamin use before pregnancy, were: 0.68 [95% confidence interval (CI), 0.46–1.00; 0.55 (95% CI, 0.32–0.93) and 0.68 (95% CI, 0.46–1.01), respectively. The ORs for use of these supplements during pregnancy were also below unity, but generally closer to the null than those for the prepregnancy period. There was some evidence of an inverse dose–response during each time period. Conclusions: These results suggest that folic acid supplements before and possibly during pregnancy may protect against CBT. Such associations are biologically plausible through established mechanisms. Impact: This study provides evidence of a specific protective effect of prenatal folic acid supplementation against the risk of CBT that is not attributable to the actions of the other micronutrients investigated. Cancer Epidemiol Biomarkers Prev; 21(11); 1933–41. ©2012 AACR.

Buccal Swabs and Treated Cards: Methodological Considerations for Molecular Epidemiologic Studies Examining Pediatric Populations

Self-collection of buccal cells provides a noninvasive method for obtaining biologic samples for genetic analyses in pediatric studies. Nevertheless, low yields, microbial contamination, and degradation of buccal samples present challenges for epidemiologic studies incorporating genetic investigations. The aims of this study were to compare the quality and yield of DNA extracted from buccal specimens with BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) or FTA cards (Whatman, Inc., Clifton, New Jersey) and to investigate the use of whole-genome amplification (WGA) for increasing DNA yields for single nucleotide polymorphism analyses. Buccal specimens were collected from 55 children with acute lymphoblastic leukemia and 52 control children without acute lymphoblastic leukemia in New South Wales, Australia, in 2003-2004. Real-time polymerase chain reaction was used to evaluate polymorphisms in the genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T). Results showed that DNA could be isolated from buccal specimens collected by use of both methods and that yields could be substantially improved with WGA without introducing genotyping error. However, DNA quality was poorer in samples collected by BuccalAmp swabs, and the presence of polymerase chain reaction inhibitors in these samples reduced the sensitivity of quantitative real-time PCR analysis. These findings show that different methods for collecting buccal samples impact on the downstream success of genetic investigations and influence DNA quality after WGA.

Childhood Acute Lymphoblastic Leukemia and Indicators of Early Immune Stimulation: A Childhood Leukemia International Consortium Study

The associations between childhood acute lymphoblastic leukemia (ALL) and several proxies of early stimulation of the immune system, that is, day-care center attendance, birth order, maternally reported common infections in infancy, and breastfeeding, were investigated by using data from 11 case-control studies participating in the Childhood Leukemia International Consortium (enrollment period: 1980-2010). The sample included 7,399 ALL cases and 11,181 controls aged 2-14 years. The data were collected by questionnaires administered to the parents. Pooled odds ratios and 95% confidence intervals were estimated by unconditional logistic regression adjusted for age, sex, study, maternal education, and maternal age. Day-care center attendance in the first year of life was associated with a reduced risk of ALL (odds ratio = 0.77, 95% confidence interval: 0.71, 0.84), with a marked inverse trend with earlier age at start (P < 0.0001). An inverse association was also observed with breastfeeding duration of 6 months or more (odds ratio = 0.86, 95% confidence interval: 0.79, 0.94). No significant relationship with a history of common infections in infancy was observed even though the odds ratio was less than 1 for more than 3 infections. The findings of this large pooled analysis reinforce the hypothesis that day-care center attendance in infancy and prolonged breastfeeding are associated with a decreased risk of ALL.

Clinical significance of minimal residual disease at day 15 and at the end of therapy in childhood acute lymphoblastic leukaemia

Detection of minimal residual disease (MRD) after induction and consolidation therapy is highly predictive of outcome for childhood acute lymphoblastic leukaemia (ALL) and is used to identify patients at high risk of relapse in several current clinical trials. To evaluate the prognostic significance of MRD at other treatment phases, MRD was measured by real‐time quantitative polymerase chain reaction on a selected group of 108 patients enrolled on the Australian and New Zealand Children’s Cancer Study Group Study VII including 36 patients with a bone marrow or central nervous system relapse and 72 matched patients in first remission. MRD was prognostic of outcome at all five treatment phases tested: at day 15 (MRD ≥ 5 × 10−2, log rank P < 0·0001), day 35 (≥1 × 10−2, P = 0·0001), 4 months (≥5 × 10−4, P < 0·0001), 12 months (MRD ≥ 1 × 10−4, P = 0·006) and 24 months (MRD ≥ 1 × 10−4, P < 0·0001). Day 15 was the best early MRD time‐point to differentiate between patients with high, intermediate and low risk of relapse. MRD testing at 12 and particularly at 24 months, detected molecular relapses in some patients up to 6 months before clinical relapse. This raised the question of whether a strategy of late monitoring and salvage therapy will improve outcome.

Parental alcohol consumption and risk of childhood acute lymphoblastic leukemia and brain tumors

PurposeChildhood acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and brain tumors (CBTs) are the leading cause of cancer death in children. In our Australian case–control studies of these cancers, we investigated whether parental alcohol consumption before or during pregnancy was associated with risk.MethodsCases were identified through the ten Australian pediatric oncology centers, and controls were recruited through national random-digit dialling. Detailed information on alcohol consumption, including beverage type, amount, and timing, was collected from 690 case families (388 ALL and 302 CBT) and 1,396 control families. Data were analyzed using unconditional logistic regression.ResultsWe found no evidence that maternal alcohol use before or during pregnancy was associated with an increased risk of either cancer; rather, there was evidence of inverse associations, particularly with wine. For both cancers, we observed U-shaped associations with paternal alcohol consumption in the year before the pregnancy, possibly driven by reduced risk at moderate levels of beer and wine intake and increased risk associated with high levels of beer intake. Moderate intake of spirits by fathers was associated with an increased risk of CBT but not ALL. These findings would be strengthened by corroboration in other studies. While the inverse associations with wine may be interesting mechanistically, the public health message remains that maternal alcohol use during pregnancy causes serious disorders in the offspring and should be avoided.ConclusionsOur findings suggest that men, as well as women, should limit their alcohol intake when planning a pregnancy.

Parental smoking and risk of childhood brain tumors

Childhood brain tumors (CBT) are the leading cause of cancer death in children, yet their etiology remains largely unknown. Tobacco smoke contains 61 known carcinogens and increases the risk of several adult cancers. This study investigated associations between parental smoking and risk of CBT in a population‐based case–control study conducted between 2005 and 2010. Cases were identified through all ten Australian pediatric oncology centers, controls via nationwide random‐digit dialing, frequency matched to cases on age, sex and state of residence. Parental smoking information was obtained for 302 cases and 941 controls through mailed questionnaires that requested average daily cigarette use in each calendar year from age 15 to the childs birth, linked to residential and occupational histories. Data were analyzed using unconditional logistic regression, adjusting for frequency matching variables and potential confounders. Overall, parental smoking before or during pregnancy showed no association with CBT risk. The odds ratios for maternal smoking before and during pregnancy were 0.99 (95% CI: 0.70, 1.40) and 0.89 (95% CI: 0.61, 1.21), respectively, and those for paternal smoking before and during pregnancy were 0.99 (95% CI: 0.71, 1.38) and 1.04 (95% CI: 0.74, 1.46), respectively. In children under 24 months of age, the odds ratios for maternal smoking preconception and during pregnancy were 5.06 (95% CI 1.35–19.00) and 4.61 (95% CI: 1.08, 19.63), although these results were based on modest numbers. Future studies should investigate the associations between maternal smoking and risk of CBT by the childs age of diagnosis.

Effect of Long-Term Infection with nef-Defective Attenuated HIV Type 1 on CD4+ and CD8+ T Lymphocytes: Increased CD45RO+ CD4+ T Lymphocytes and Limited Activation of CD8+ T Lymphocytes

Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/LTR mutation and have maintained relatively stable CD4+ T lymphocyte counts for 14-18 years. Flow cytometric analysis was used to examine the phenotype of CD4+ and CD8+ T lymphocytes in these subjects, including the immunologically important naive (CD45RA+CD62L+), primed (CD45RO+), and activated (CD38+HLA-DR+ and CD28-) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4+ and CD8+ T lymphocytes, but had primed CD45RO+ CD4+ T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8+ T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8+ T lymphocytes. Therefore, the result of long-term infection with natural nef/LTR mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4+ T lymphocyte subsets in vivo, and minimal activation of CD8+ T lymphocytes.