Epidemiological evidence suggests that patterns of infection after birth have a causal role in triggering ALL. The delayed infection hypothesis lends itself to epidemiological evaluation in a case–control setting. A prediction of the model was that common infections in infancy should be protective against BCP-ALL. There is no prior reason to implicate any particular infectious agent (for example, bacteria, virus or parasite), and the relevant infections need not be symptomatic or pathological. A longstanding need for microbial, immune network modulation might reflect common, commensal, or ‘old friend’ organisms, such as gut microbiota, soil mycobacteria or helminth parasites93. In this context, a surrogate of overall infectious exposure during infancy could be considered an appropriate variable. Quantifiable surrogates include social exposures of infants in the home, related to the number of siblings and birth order, or in day care centres, and breastfeeding. These variables have been investigated in epidemiological case–control or cohort studies for risk associations with ALL overall and, in some instances, selectively for the major BCP-ALL subset.

Impact of day care attendance in infancy

In the 1990s, the UK Children’s Cancer Study Group (UKCCS) was set up to test the delayed infection hypothesis, in a case–control context, in addition to analysing other exposures including ionizing and non-ionizing (such as that from electromagnetic fields) radiation and chemicals8. Day care attendance was chosen as one surrogate for infectious exposure because this was well documented as a context for increased social contacts facilitating spread of common infections94. The UKCCS involved almost 1,300 patients with ALL (all subtypes) and over 6,000 matched controls. Although only a relatively small number of controls experienced day care in the first 12 months of life, the data showed a significant protective impact on risk of ALL overall and on BCP-ALL95.

This association has been documented in additional studies in California96, Scandinavia97 and France98 and in an international consortium99, and has been endorsed by a meta-analysis100. The meta-analysis noted significant between study heterogeneity, and one early, large-scale study failed to detect any impact of day care on risk of ALL101. No protection is afforded against childhood AML by day care attendance or, to date, any other paediatric cancer, which increases the confidence that the associations seen in ALL were not confounded by social or other variables.

It was anticipated that assessing, in a case–control fashion, actual infections in infancy would be informative. This, however, is fraught with difficulties and has provided mixed results. Parental recall is known to be suspect or inaccurate in this respect102. Medical records are more reliable, particularly in the UK, with nationwide registration of children with general practitioners and a free health service. One such analysis found more, rather than fewer, infections reported for children who subsequently developed ALL than for controls102. The main difficulty here, other than possible bias in use of general practitioner services, is that we do not know whether the relevant modulating infectious exposures in infancy are necessarily symptomatic; they might well not be. In this sense, the surrogate of day care could be considered preferable. However, several studies have reported, in accord with the hypothesis, an inverse relationship between common infections in early life, including inner ear infections, and risk of ALL98,101,103,104,105,106.

Birth order and risk of ALL

A further surrogate measure of infectious exposures in infancy is the number of siblings cohabiting and, in particular, birth order. The prediction was that firstborns would be more at risk than laterborns, who would, as infants, benefit from protective exposures via older siblings. One large UK-based study (with >3,000 patients with ALL and the same number of matched controls) found a striking association of birth order with risk of ALL, but not ofAML107. Other case–control studies in France98 and California100 also found a significantly increased risk of ALL for firstborn (versus thirdborn) children, as did a recent international cohort analysis (O. Paltiel, personal communication).

If natural infections early in life reduce risk of ALL, then it might be expected that some vaccinations would have an effect. The data on vaccination histories have produced null or inconsistent results. However, there is one exception: immunization against Haemophilus influenzae type B in infancy appears to confer a degree of protection against ALL108.

If the natural microbiota is part of a longstanding and critical interaction with the developing immune system, then antibiotic use in infancy might increase risk of ALL. This has not been systematically evaluated to date, though an earlier report from China did suggest an increased risk associated with exposure to chloramphenicol109.

Mode of delivery, breastfeeding and risk of ALL

The mode of delivery at birth influences the early exposure of newborn babies to benign microbiota110, as caesarean delivery deprives newborn babies of the microbial exposures associated with vaginal passage. Cohort and case–control studies have reported a significantly increased risk of ALL associated with caesarean delivery111,112,113,114. No such increased risk was observed for brain cancer or lymphoma111.

Breastfeeding during infancy provides nutritional support, maternal antibodies, anti-inflammatory molecules, some maternal cells, microorganisms (lactobacilli) and oligosaccharides that nourish the infant’s intestinal microbiome (Bifidobacteria spp.)115. It might be anticipated that long-term breastfeeding would have a modulating effect on the immune system of infants and reduce the risk of ALL. Seventeen case–control studies of the impact of breastfeeding on ALL risk have been published116. In the largest of these, from the USA117 and UK118, there was a reduced risk of ALL (10–20%) associated with breastfeeding of 6 months or more. Five meta-analyses have now been published with concordant conclusions, and the latest of these indicated a reduced ALL risk of ~20% for breastfeeding of 6 months or more116.

Clusters of ALL

Although no specific microbial agent or a unique transforming virus is suspected in ALL, there might be one circumstance where a single type of infection is involved: the very rare cases of space–time clusters. A prediction of the hypothesis would be that a single cluster of affected patients might be associated with a single infection or microorganism species, but independent, space–time clusters could involve different infectious triggers.

Many putative clusters of childhood leukaemia have been reported, but two stand out. The first was in Niles, a suburb of Chicago, Illinois, USA, in 1957–1960 — where there were eight patients (relative risk (RR) 4.3) diagnosed with ALL or ‘stem cell’ leukaemia119. All patients and/or their older siblings attended the same school. The second cluster involved 13 patients with BCP-ALL over 4 years (2000–2004; RR 12.0), but 10 of those patients were diagnosed within just 10 months in 2001, in the small town of Fallon, Nevada, USA120.

A neglected aspect of these two clusters is that the patients, though resident in the cluster area at the time of diagnosis, were mostly born outside of that area7. Additionally, the clusters involved children diagnosed with ALL at different ages (2–11 years) and a narrow time frame of diagnoses. Given what we now know of the natural history of ALL, these data then indicate that any causal exposure linked to the cluster would have to be proximal in time to diagnosis (rather than prenatal) and, therefore, promotional. The Niles cluster was linked, observationally, to an outbreak of streptococcal fever119. The cause(s) of the Fallon cluster of ALL remain unresolved and contentious, though a possible role of adenovirus was hypothesized120.

A significant space–time cluster of BCP-ALL in Milan, Italy has recently been recorded121. Seven patients were diagnosed in a 4-week period; four of these lived within one small residential area, and three of these four attended a single school. The Kulldorff scan method122 identified this as a significant space–time cluster (P = 0.017). Given the narrow time window of the diagnoses (4 weeks) and the age range of the patients (2–11 years), any causal exposure, as in Niles and Fallon, would be proximal to diagnosis, promoting overt ALL evolution from a prior and covert pre-leukaemic state. The Milan cases sparked substantial public anxiety, particularly in relation to the school, and a detailed epidemiological investigation was launched. No link was found with ionizing radiation, non-ionizing radiation or chemicals. There was, however, an association with a particular common infection. All seven patients had been infected with endemic influenza A H1N1 swine flu virus during the epidemic that preceded the ALL cluster by 3–6 months. The infection frequency in children in Milan during the same period was relatively high, at around one-third, but this still indicated that the link with patients was significantly different from expected (P = 0.01)121. Six of the seven patients were firstborn children, and none attended day care in the first year of life.

Proof of a causal role for infections in these situations is not possible, and clustering of cases by chance cannot be excluded. But the observations accord with predictions of the infection hypothesis and highlight that influenza viruses are potential promoting agents for ALL. A previous study in the UK observed peaks in the incidence of childhood ALL ~6 months after seasonal influenza epidemics123. A final piece of epidemiological evidence indirectly supporting a role of common infection in childhood ALL comes from anecdotal but striking observations of rapid changes in the incidence of ALL that were preceded by major social changes in Germany and Hong Kong (Supplementary Box 2).

There is no compelling reason for postulating an exclusive role for influenza viruses or, indeed, for viruses. A role for cytomegalovirus (CMV) in ALL has been proposed but as an early, in utero modulator of immunity rather than as a proximal trigger124.

In some respects, it is surprising that the epidemiological data are as consistent as they are for individual factors related to infection in ALL, because many variables will interact to influence patterns of microbial exposures in early life (Supplementary Box 3).