Our data provide evidence of a potential link between history of a viral illness and a DNA methylation pattern in ALL cells. The DNA methylation pattern that was linked with higher levels of PVB19 IgG is a combination of both increased and decreased DNA methylation at specific CpGs compared with other ALLs of the same immunophenotype but different DNA methylation patterns. This observation was replicated for specific individual genes, but future replications using different populations and technologies will be necessary to further characterize and confirm this phenomenon. Kerr and colleagues25
proposed that infection with PVB19 was involved in the onset of ALL, although the virus was not in blood circulation at the time of diagnosis. These authors also showed a prominent presence of PVB19 DNA in the cerebrospinal fluid of patients with ALL when compared with with normal controls (p = 0.046).25
Heegaard et al. proposed that PVB19 infection could be the cause of cytopenic episodes occasionally preceding an ALL diagnosis, this relationship being possibly etiologic.26
Patients with weak erythropoiesis typically experience a transient aplastic crisis and anemia with PVB19 infection; viral clearance is associated with positive regulation and regrowth of the bone marrow.27
This presents a possible scenario where viral damage impacting a DNA methylation pattern is followed by enhanced proliferation and fixation of that pattern. An alternative explanation for the DNA methylation pattern associated with the virus could be the creation of a permissive environment for the growth of cells with a particular phenotype that is reflected in a common DNA methylation pattern. Either of these mechanisms could be considered a “bystander” effect, with the primary infected cells (erythroblasts) not the target cells for cancer. Precursor-B cells could be affected by a cytokine “storm” during an infection, as well as being exposed to toxic reactive oxygen species in the clearance of infection. Developing B cells have a capacity for rapid expansion and proliferation and exhibit a limited repertoire of genetic changes such as chromosomal translocations and gene deletions, which may be promoted by localized inflammation in the bone marrow following an infection.
Our result is consistent with a single prior study measuring the presence of PVB19 and DNA methylation in diagnostic samples. While “history” of PVB19 infection was not measured, Yalcin and colleagues noted a significant association between the concurrent presence of PVB19 DNA in diagnostic samples and methylation in the p15 gene promoter in adult leukemia patients.28
Future studies should incorporate measures of both current and historic infections.
The proposed role of viruses in childhood ALL can seem somewhat paradoxical. Epidemiological studies have long portrayed a protective role for childhood ALL concerning infections, as surrogate markers for infection such as early daycare seem to be protective.10
However, additional studies indicate that infections severe enough to result in a physician visit may be a risk factor.29,30
While exposure to a variety of antigens may be protective via a normal modulation and development of the immune system, a strong reaction to infections agents may induce widespread immune stimulation as well as specific anti-viral defenses that may impact leukemogenesis, in particular for a virus with bone marrow tropism like PVB19. Children who contract leukemia tend to have pre-leukemic mutations, present in many cases prior to birth.31–33
A strong reaction in the vicinity of a pre-cancerous cell may produce a characteristic immune reaction that results in the homogeneous DNA methylation pattern observed among some DNA methylation classes, in particular class “LRR” () whose children harbor the highest PVB19 IgG levels. It should be noted that this methylation class does not harbor an overall “more methylated” phenotype, only a different pattern of DNA methylation compared with other childhood ALLs from this series.
Epigenetic traits (e.g., aberrant DNA methylation) are known to be related to ALL pathogenesis along with genetic variations such as translocation and altered chromosome number. There are few reports, however, analyzing methylation profile in childhood lymphocytic leukemias.34–38
Most of them analyze a small numbers of genes except for one, where the authors used a customized platform by Illumina with probes for 386 genes.35
Our current study is the only one we are aware of that shows any relation of DNA methylation in ALL to infections.
We chose genes to replicate based only on their strong associations in the GoldenGate panel, but do note that they are derived from a “Cancer Panel” and may have an impact on leukemogenesis. All three genes also have key roles in hematopoietic and immune function as well. LCK and LTA are both expressed in lymphocytes and are critical immune regulatory genes. LCK is a T-cell specific tyrosine kinase, and LTA encodes a cytokine produced by lymphocytes that is highly induced during viral infection. DAPK is a serine-threonine kinase that is a positive mediator of interferon-associated cell death. Future studies should explore a potential for mechanistic links between viral infection and DNA methylation of key immune genes with subsequent impact on leukemogenesis.
Finally, our analysis here does not implicate PVB19 as a “causative agent” in leukemogenesis nor does it satisfy Koch's postulates. We do provide evidence for a potential new role for a common virus in leukemogenesis; a virus may impact the epigenetic state of a leukemia clone in a specific manner. The findings have implications on infections impacting leukemia risk, and may impact current etiologic models of childhood leukemogenesis involving infection. Further research should examine the possible role of other infectious agents on DNA methylation patterns as well as tease out the mechanistic relationship between Parvovirus B19 and DNA methylation in leukemia cells. Such future investigations should also investigate the role of PVB19 infection on the DNA methylation patterns of normal cells, sorted into homogenous populations, including both the direct targets (erythroblasts) and bystander cells.