Stephen S. Francis

Caesarean delivery and risk of childhood leukaemia: a pooled analysis from the Childhood Leukemia International Consortium (CLIC)

BACKGROUND Results from case-control studies have shown an increased risk of acute lymphoblastic leukaemia (ALL) in young children born by caesarean delivery, and prelabour caesarean delivery in particular; however, an association of method of delivery with childhood leukaemia subtypes has yet to be established. We therefore did a pooled analysis of data to investigate the association between childhood leukaemia and caesarean delivery. METHODS We pooled data from 13 case-control studies from the Childhood Leukemia International Consortium done in nine countries (Canada, Costa Rica, Egypt, France, Germany, Greece, Italy, New Zealand, and the USA) for births from 1970-2013. We analysed caesarean delivery overall and by indications that probably resulted in prelabour caesarean delivery or emergency caesarean delivery. We used multivariable logistic regression models, adjusted for childs birthweight, sex, age, ethnic origin, parental education, maternal age, and study, to estimate odds ratios (ORs) and 95% CIs for the risk of ALL and acute myeloid leukaemia (AML) in children aged 0-14 years at diagnosis. FINDINGS The studies provided data for 8780 ALL cases, 1332 AML cases, and 23 459 controls, of which the birth delivery method was known for 8655 (99%) ALL cases, 1292 (97%) AML cases, and 23 351 (>99%) controls. Indications for caesarean delivery were available in four studies (there were caesarean deliveries for 1061 of 4313 ALL cases, 138 of 664 AML cases, and 1401 of 5884 controls). The OR for all indications of caesarean delivery and ALL was 1·06 (95% CI 0·99-1·13), and was significant for prelabour caesarean delivery and ALL (1·23 [1·04-1·47]; p=0·018). Emergency caesarean delivery was not associated with ALL (OR 1·02 [95% CI 0·81-1·30]). AML was not associated with caesarean delivery (all indications OR 0·99 [95% CI 0·84-1·17]; prelabour caesarean delivery 0·83 [0·54-1·26]; and emergency caesarean delivery 1·05 [0·63-1·77]). INTERPRETATION Our results suggest an increased risk of childhood ALL after prelabour caesarean delivery. If this association is causal, maladaptive immune activation due to an absence of stress response before birth in children born by prelabour caesarean delivery could be considered as a potential mechanism. FUNDING National Cancer Institute.

Mode of Delivery and Risk of Childhood Leukemia

Background: Childhood infection and immune response have long been suspected in the etiology of childhood leukemia, specifically acute lymphoblastic leukemia (ALL). Normal primary inoculation of the core human microbiome is circumvented by cesarean section (CS) delivery, which is a proposed modulator of both immune response and early-life infection. Methods: In this study, we examined CS delivery and the risk of childhood leukemia using data from the California Childhood Leukemia Study (CCLS) case–control study and additive logistic regression models. Results: We observed no association between CS and acute myelogenous leukemia [OR, 0.96; 95% confidence interval (CI), 0.52–1.55]. We observed a suggestive association for ALL and CS (OR, 1.22; 95% CI, 0.97–1.54). When examining common ALL (cALL), defined as ALL with expression of CD10 and CD19 surface antigens and diagnosis occurring between 2 and 5.9 years of age, we found a significant association with CS (OR, 1.44; 95% CI, 1.0–2.06). ALL subjects that are not cALL showed a similar risk as ALL overall (OR, 1.15; 95% CI, 0.91–1.44). Because of previous findings suggesting effect modification, we stratified cALL subjects by Hispanic status. Although we observed no relationship for CS in non-Hispanics (OR, 1.14; 95% CI, 0.72–1.79), we did observe a strong association between cALL and CS in Hispanics (OR, 2.34; 95% CI, 1.23–4.46). Conclusion: Within the CCLS, CS delivery seems to be associated with cALL and Hispanic subjects may be driving the association. Impact: Further research combined with investigations into response to early infection and the microbiome is warranted. Cancer Epidemiol Biomarkers Prev; 23(5); 876–81. ©2014 AACR.

In utero cytomegalovirus infection and development of childhood acute lymphoblastic leukemia

It is widely suspected, yet controversial, that infection plays an etiologic role in the development of acute lymphoblastic leukemia (ALL), the most common childhood cancer and a disease with a confirmed prenatal origin in most cases. We investigated infections at diagnosis and then assessed the timing of infection at birth in children with ALL and age, gender, and ethnicity matched controls to identify potential causal initiating infections. Comprehensive untargeted virome and bacterial analyses of pretreatment bone marrow specimens (n = 127 ALL in comparison with 38 acute myeloid leukemia cases in a comparison group) revealed prevalent cytomegalovirus (CMV) infection at diagnosis in childhood ALL, demonstrating active viral transcription in leukemia blasts as well as intact virions in serum. Screening of newborn blood samples revealed a significantly higher prevalence of in utero CMV infection in ALL cases (n = 268) than healthy controls (n = 270) (odds ratio [OR], 3.71, confidence interval [CI], 1.56-7.92, P = .0016). Risk was more pronounced in Hispanics (OR=5.90, CI=1.89-25.96) than in non-Hispanic whites (OR=2.10 CI= 0.69-7.13). This is the first study to suggest that congenital CMV infection is a risk factor for childhood ALL and is more prominent in Hispanic children. Further investigation of CMV as an etiologic agent for ALL is warranted.

Bacterial microbiome of breast milk and child saliva from low-income Mexican-American women and children

Background:The childhood salivary microbiome, which plays an important role in healthy development, may be influenced by breast milk consumption. The composition of the milk microbiome and the role it plays in the establishment of the infant microbiome are not well understood.Methods:Here, we sequenced the bacterial 16S rRNA gene to characterize microbial communities in breast milk and 5-year-old child saliva from 10 low-income, Mexican-American mother–child pairs with a high prevalence of obesity.Results:Members of the genus Streptococcus dominated both milk and salivary microbial communities in most subjects. Staphylococcus was observed predominately in milk samples while Prevotella was more prevalent in child saliva. No statistically significant relationships were observed between maternal and child microbiomes or between child microbiome and BMI. However, prepregnancy BMI was correlated with both lower Streptococcus abundance (r = −0.67) and higher microbial diversity (r = 0.77) in breast milk (P < 0.05 for both). Diversity estimates were notably similar to data from other low-income cohorts or children.Conclusion:These findings contribute to the currently limited state of knowledge regarding the breast milk and salivary microbiomes in mother–child pairs and may inform future studies seeking to elucidate the relationship between early-life microbial exposures and pediatric health.

Common genetic variants associated with telomere length confer risk for neuroblastoma and other childhood cancers.

Aberrant telomere lengthening is an important feature of cancer cells in adults and children. In addition to somatic mutations, germline polymorphisms in telomere maintenance genes impact telomere length. Whether these telomere-associated polymorphisms affect risk of childhood malignancies remains largely unexplored. We collected genome-wide data from three groups with pediatric malignancies [neuroblastoma (N = 1516), acute lymphoblastic leukemia (ALL) (N = 958) and osteosarcoma (N = 660)] and three control populations (N = 6892). Using case-control comparisons, we analyzed eight single nucleotide polymorphisms (SNPs) in genes definitively associated with interindividual variation in leukocyte telomere length (LTL) in prior genome-wide association studies: ACYP2, TERC, NAF1, TERT, OBFC1, CTC1, ZNF208 and RTEL1 Six of these SNPs were associated (P < 0.05) with neuroblastoma risk, one with leukemia risk and one with osteosarcoma risk. The allele associated with longer LTL increased cancer risk for all these significantly associated SNPs. Using a weighted linear combination of the eight LTL-associated SNPs, we observed that neuroblastoma patients were predisposed to longer LTL than controls, with each standard deviation increase in genotypically estimated LTL associated with a 1.15-fold increased odds of neuroblastoma (95%CI = 1.09-1.22; P = 7.9×10(-7)). This effect was more pronounced in adolescent-onset neuroblastoma patients (OR = 1.46; 95%CI = 1.03-2.08). A one standard deviation increase in genotypically estimated LTL was more weakly associated with osteosarcoma risk (OR = 1.10; 95%CI = 1.01-1.19; P = 0.017) and leukemia risk (OR = 1.07; 95%CI = 1.00-1.14; P = 0.044), specifically for leukemia patients who relapsed (OR = 1.19; 95%CI = 1.01-1.40; P = 0.043). These results indicate that genetic predisposition to longer LTL is a newly identified risk factor for neuroblastoma and potentially for other cancers of childhood.

GWAS in childhood acute lymphoblastic leukemia reveals novel genetic associations at chromosomes 17q12 and 8q24.21

Childhood acute lymphoblastic leukemia (ALL) (age 0–14 years) is 20% more common in Latino Americans than non-Latino whites. We conduct a genome-wide association study in a large sample of 3263 Californian children with ALL (including 1949 of Latino heritage) and 3506 controls matched on month and year of birth, sex, and ethnicity, and an additional 12,471 controls from the Kaiser Resource for Genetic Epidemiology Research on Aging Cohort. Replication of the strongest genetic associations is performed in two independent datasets from the Children’s Oncology Group and the California Childhood Leukemia Study. Here we identify new risk loci on 17q12 near IKZF3/ZPBP2/GSDMB/ORMDL3, a locus encompassing a transcription factor important for lymphocyte development (IKZF3), and at an 8q24 region known for structural contacts with the MYC oncogene. These new risk loci may impact gene expression via local (four 17q12 genes) or long-range (8q24) interactions, affecting function of well-characterized hematopoietic and growth-regulation pathways.Childhood acute lymphoblastic leukemia is common in Latino Americans. Here, the authors conduct a genome-wide association study in a Californian cohort containing children of Latino heritage, and identify loci on 17q12 and 8q24 which may affect hematopoietic and growth-regulation pathways.

Cesarean Section and Risk of Childhood Acute Lymphoblastic Leukemia in a Population-Based, Record-Linkage Study in California

The relationship of mode of delivery to risk of childhood acute lymphoblastic leukemia (ALL) is uncertain. After linking birth records and cancer registry data from California, we conducted a population-based case-control study to investigate the role of delivery by cesarean section (C-section) in the etiology of childhood ALL. This study included 5,081 cases and 18,927 matched controls born in 1978–2009; more detailed data were available on type of C-section (i.e., elective vs. emergency) for a subset of 1,552 cases and 5,688 controls. No association was observed between C-section overall and childhood ALL risk (<15 years of age), but elective C-section was associated with a significantly elevated risk of ALL (odds ratio (OR) = 1.17, 95% confidence interval (CI): 1.01, 1.36). At the peak ages of ALL incidence (2–4 years), C-section was associated with an 11% higher risk of ALL (OR = 1.11, 95% CI: 1.01, 1.22) compared with vaginal delivery, and the magnitude of the association was larger for elective C-section (OR = 1.38, 95% CI: 1.11, 1.70). Emergency C-section was not associated with childhood ALL. Because of design features minimizing nonparticipation and inaccurate recall, this record linkage–based study is less prone to bias. Our results suggest that delivery by elective C-section was associated with a higher risk of childhood ALL, especially at the peak ages of incidence. It is important to evaluate possible mechanisms, because this potential risk factor is modifiable.

A germ-line deletion of APOBEC3B does not contribute to subtype-specific childhood acute lymphoblastic leukemia etiology

Approximately 4/100,000 children are diagnosed with acute lymphoblastic leukemia (ALL) in the United States annually. Early life exposures related to immune priming (i.e. vaginal birth, daycare attendance, and high birth order) and having fewer infections requiring medical treatment have been inversely associated with disease, suggesting an etiological role of infectious agents, perhaps via dysregulation of the immune system. Patterns of somatic mutation in ALL tumors give further insight into disease etiology. An innate immune enzyme, APOBEC3B (Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B), inhibits viral infection by inducing TpC>T nucleotide changes in foreign nucleic acid. This mutation signature has been identified in tumor genomes of several cancer types with known (cervical, head and neck, stomach) or hypothesized (breast) infectious etiologies, and is attributed to aberrant enzymatic activity of APOBEC3B. The APOBEC3B point mutation signature is also predominant in ALL, but with subtype specificity. The signature is present in ETV6-RUNX1 fusion ALL but absent in high hyperdiploid ALL. The high expression of APOBEC3B in lymphoblasts further justifies examination of functional APOBEC3B polymorphisms in ALL etiology. An ~30kb germline deletion polymorphism at the APOBEC3B locus has been associated with increased risk of several cancers that bear the APOBEC3B mutation signature and studies have shown that the deletion transcript yields an enzyme with a higher in vitro propensity for collateral genomic DNA damage than its wild-type counterpart. In fact, the signature TpC>T point mutation burden is higher in the tumors of ALL and breast cancer patients carrying the germline APOBEC3B deletion compared with those without. The deletion is common in populations of Native American ancestry (approx. 60%), and relatively rare in Europeans and Africans (6% and 0.9%, respectively). Hispanic children, whose genetic ancestry is typically comprised of a mixture of Native American, European, and African ancestries, are at the greatest risk for developing ALL in the United States. While it has been suggested that the APOBEC3B deletion polymorphism contributes to the patterns of somatic mutations observed in ALL tumors, it is not known whether the variant contributes to disease risk. Here, we report results from the first association study of germline APOBEC3B variants in childhood ALL risk. The APOBEC3B deletion genotype was assessed in California Childhood Leukemia Study (CCLS) case and control subjects (see Online Supplementary Methods for enrollment details) with a polymerase chain reaction (PCR)-based assay (n=1126). The deletion was tested for association with childhood ALL status overall and within ETV6-RUNX1 fusion and high hyperdiploid ALL subtypes. Overall, controls tended to be wealthier than cases with a higher proportion self-reporting as white and non-Hispanic (Table 1). APOBEC3B deletion copy number was detected using a validated polymerase chain reaction (PCR) method described previously. A total of 518 ALL cases and 608 controls were genotyped using this PCR assay, with copy number (homozygote wild-type, heterozygote, and homozygote deletion) determined from agarose gel electrophoresis results (Online Supplementary Figure S1). A χ test for Hardy-Weinberg equilibrium was performed; the null hypothesis of deletion equilibrium was accepted among controls after stratifying by Hispanic versus nonHispanic ethnicity (P=0.45 and 0.45, respectively). Ethnic heterogeneity in the CCLS population is supported by the distribution of multidimensional scaling (MDS) components compared to reference populations (Online Supplementary Figure S2). After adjusting for global genetic ancestry (first 3 MDS components), no association was observed between the APOBEC3B deletion and overall ALL risk for the additive, dominant, or recessive models, nor after stratification by cytogenetic subtype (Table 2). When study subjects were stratified by self-reported Hispanic status, results did not change (Online Supplementary Table S1). Previous studies have identified SNPs within the APOBEC3 region that are associated with cancer risk independent of the APOBEC3B deletion. Thus, we tested all SNPs across the APOBEC3 gene region (chr22:39,200,00039,650,000) that passed quality filtering (8275 SNPs) for association with ALL in 1083 cases and 1137 controls. After controlling for genetic ancestry, no variant reached statistical significance after correcting for multiple testing (Online Supplementary Figure S3). A SNP ~20Kb upstream of APOBEC3B and previously associated with bladder cancer, rs1014971, was not associated with ALL risk [Odds Ratio (OR) 0.91, P=0.33]. The top association was seen for rs73424730 (OR 1.35, P=0.004), a SNP ~ 100Kb downstream of the APOBEC3H gene.

Genotyping Oral Commensal Bacteria to Predict Social Contact and Structure.

Social network structure is a fundamental determinant of human health, from infectious to chronic diseases. However, quantitative and unbiased approaches to measuring social network structure are lacking. We hypothesized that genetic relatedness of oral commensal bacteria could be used to infer social contact between humans, just as genetic relatedness of pathogens can be used to determine transmission chains of pathogens. We used a traditional, questionnaire survey-based method to characterize the contact network of the School of Public Health at a large research university. We then collected saliva from a subset of individuals to analyze their oral microflora using a modified deep sequencing multilocus sequence typing (MLST) procedure. We examined micro-evolutionary changes in the S. viridans group to uncover transmission patterns reflecting social network structure. We amplified seven housekeeping gene loci from the Streptococcus viridans group, a group of ubiquitous commensal bacteria, and sequenced the PCR products using next-generation sequencing. By comparing the generated S. viridans reads between pairs of individuals, we reconstructed the social network of the sampled individuals and compared it to the network derived from the questionnaire survey-based method. The genetic relatedness significantly (p-value < 0.001) correlated with social distance in the questionnaire-based network, and the reconstructed network closely matched the network derived from the questionnaire survey-based method. Oral commensal bacterial are thus likely transmitted through routine physical contact or shared environment. Their genetic relatedness can be used to represent a combination of social contact and shared physical space, therefore reconstructing networks of contact. This study provides the first step in developing a method to measure direct social contact based on commensal organism genotyping, potentially capable of unmasking hidden social networks that contribute to pathogen transmission.

Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8×10−4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches.