Evaluation of the Association Between Congenital Cytomegalovirus Infection and Pediatric Acute Lymphoblastic Leukemia

This case-control study evaluates the association between congenital cytomegalovirus infection and pediatric acute lymphoblasic leukemia.


Introduction
Acute lymphoblastic leukemia (ALL) is the most common form of pediatric cancer, accounting for nearly 20% of all malignant neoplasms diagnosed in persons under 20 years of age. 1,2Highly penetrant genetic predispositions, such as Down syndrome, cause ALL in less than 5% of cases. 3vironmental risk factors have also been suggested, such as exposure to ionizing radiation, but the evidence is not conclusive. 4,5Largely, the cause of most ALL cases remains unknown.
Recently, cytomegalovirus (CMV) has emerged as a potential risk factor of ALL.CMV is a member of the herpesvirus family (HHV 5) and is capable of transplacental infection during pregnancy.In a report by Francis et al, 6 congenital CMV (cCMV) infection was assessed in a population-based sample of newborn dried blood spots (DBS) from 268 ALL cases and 270 controls in California.Overall, cCMV was detected in 9.7% of ALL cases but only 3.0% of controls for a highly significant odds ratio (OR) of 3.71 (95% CI, 1.71-8.95).In a second study by Wiemels et al, 7 congenital and early life, clinically recognized, CMV infection and subsequent ALL were investigated in population-based registries of Sweden.The hazard ratio (HR) of hematologic malignant neoplasm among children with any medically documented cCMV or early life acquired CMV was 11.2 (95% CI, 5.8-21.5).
Together, the 2 prior studies suggest prenatal CMV infection substantially increases risk of childhood ALL.If true, congenital and early life acquired CMV infection could represent the first modifiable risk factor for childhood ALL.As universal newborn screening for cCMV is in development, it is important to establish through replication whether CMV infection at birth is a risk factor for ALL.
Therefore, this study assessed the association between cCMV and risk of childhood ALL.

Selection of Cases and Controls and Data Collection
Congenital CMV infection was assessed in a population-based case-control study through the Michigan BioTrust for Health (MBH).MBH is a Michigan Department of Health and Human Services program that oversees the use of residual newborn DBS collected shortly after birth along with routine linkage of the DBS repository to the birth and cancer registries.Cases consisted of children 0 to 14 years of age between 1987 and 2014 with an ALL diagnosis (International Classification of Diseases for Oncology, third edition code morphology 9835, 9836, and 9820) identified through the Michigan Cancer Surveillance Program and born in Michigan on or after October 1, 1987.Cytogenetic and molecular data on cases was abstracted through collaboration with 7 major Michigan children's hospitals through an instrument programmed in REDCap.Controls were selected by MBH and matched 4:1 on year of birth, sex, and mother's reported race and ethnicity.Sex and mother's race and ethnicity were matched for efficient control of confounding while year of birth was matched to have equivalent windows of exposure.Birth characteristic data and mother's race and ethnicity were obtained from linkage to the birth registry and included previously identified risk factors. 8,9Due to data suppression rules by MBH, any potential identifiable health information, such as birth year or mother's and father's age were categorized.The study was approved by the institutional review boards of the University of Minnesota and the Michigan BioTrust for Health.Parents of children with available DBS provided informed consent to MBH for the use of DBS in research studies.Informed consent was not required for this study as no cases or controls were contacted for additional information.This study is reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for observational studies.

DNA Extraction and CMV Assay
Detection of CMV DNA in DBS was performed as described elsewhere, 10 with some modifications.One 6-mm punch, equivalent to 28.27 2 mm in area, from the newborn DBS card was provided by MBH.DNA was extracted using the GenTegra GenSolve DNA Complete kit (GenTegra LLC).Briefly, Downloaded From: https://jamanetwork.com/ on 01/13/2023 the 6 mm punch was mixed with 609 μL lysis solution (GenTegra) and 11 μL of proteinase K (20 mg/mL) and incubated at 56 °C for 1.5 hours with agitation at 1400 rpm.Samples were transferred to a spin basket and centrifuged with a Recovery Solution (GenTegra).This was followed by DNA purification following the manufacturer's protocol (GenTegra).Samples were eluted in 50 μL of elution buffer and stored at −20 °C.

JAMA Network Open | Hematology
Quantitative multiplex PCR was performed as described elsewhere.The mean (SD) weight gain during pregnancy was similar across mothers of cases (31.4 [13.9]   pounds) and controls (31.0 [13.4] pounds) (t = −0.85,df = 5503; P = .39)(Table 1).There were no significant differences between mothers in smoking or alcohol use during pregnancy as recorded on birth certificates (χ 2 2 = 0.08; P = .96and χ 2 2 = 0.21; P = .90,respectively).There was a slightly higher prevalence of prepregnancy or gestational diabetes among mothers of cases (49 participants [4.1%]) than controls (153 participants [3.2%]), but this difference was not significant (χ 2 1 = 2.37; P = .12).Among controls, there was a slightly higher prevalence of uterine or vaginal bleeding during pregnancy (61 participants [1.3%]) than cases (7 participants [0.6%]) (χ 2 1 = 4.05; P = .04).Mode of delivery and plurality of birth was similar across cases and controls (χ 2 4 = 3.10; P = .54and χ We detected cCMV DNA in 6 of 1189 of ALL cases (0.5%) and 21 of 4756 of controls (0.4%) (Table 2).The crude odds of cCMV infection were not statistically different between ALL cases and controls (OR, 1.14; 95% CI, 0.46-2.83).In the multivariable model, the odds of cCMV exposure did not differ between cases compared with controls (adjusted OR, 1.30; 95% CI, 0.52-3.24).Among B-ALL cases, the odds of cCMV appeared elevated compared with matched controls; however, this point estimate was also measured imprecisely (OR, 4.0; 95% CI, 0.56-28.40).There were no cCMV positive cases among recognized T-ALL.There were 2 hyperdiploid cases among those with subtype data that were cCMV positive.When we compared CMV prevalence among hyperdiploid ALL cases and their matched controls, the model did not converge due to lack of exposure among the matched controls.However, compared with all controls (4756 participants) in an unmatched analysis, hyperdiploid ALL cases (74 cases) were 6.26 times more likely to be CMV positive (95% CI, 1.44-27.19).We also assessed this outcome in an unmatched analysis including all matched controls of cases who had subtype data available (2144 participants) and found that the odds of cCMV exposure were 13.4 times greater among hyperdiploid cases compared with controls (95% CI, 1.25-83.21).In the stratified analysis of CMV infection, we did not detect any significant associations (Table 3).Among infants whose mothers had some education beyond high school, the prevalence of CMV was significantly different across cases and controls (Fisher exact P = .04);however, the crude OR was not significant (OR, 4.97; 95% CI, 0.84-34.07).We did not detect any differences in the odds of CMV infection by mother's race and ethnicity, as all cases (6 participants) had White mothers.

Discussion
Congenital CMV infection has emerged as a potential modifiable risk factor of pediatric ALL.In response to the 2 extant studies of the topic by Francis et al 6 and Wiemels et al, 7 we conducted a large, population-based case-control study of cCMV infection and pediatric ALL.In the entire study of 1189 ALL cases and 4756 controls we did not detect an association between leukemia and exposure to cCMV infection in the main analysis.However, among hyperdiploid ALL cases, the odds of being cCMV positive were 6 times greater than unmatched controls.3][14][15] Although none of these hypotheses anticipated an infection, such as CMV, which modulates the immune system for future infections, among existing hypotheses CMV best fits the criteria of Smith's, which states the infectious agent causing ALL should possess: (1) ability to induce genomic instability; (2) specific effects on B lymphocytes; (3) higher rates of infection in lower socioeconomic status regions; (4) limited general oncogenic potential; (5) minimal symptoms associated with primary infection; and (6) ability to cross the placenta and infect the fetus, but not cause severe abnormalities. 15V is capable of causing direct chromosomal breakage in congenital infection, which is likely associated with its teratogenic properties, 16 and encodes several proteins that modulate cell cycle control and the host DNA damage response. 17The association between cCMV and B lymphocytes are CD34 + cells, which are early hematopoietic progenitor cells in bone marrow, 18,19 and the cell type in which CMV establishes latency.A study by Albano et al 20 investigated the impact of cCMV on hematopoietic progenitor cell concentrations in cord blood and found among infants with cCMV infection, CD34 + cell populations were roughly 2.6 times greater than those of matched controls.
This suggests a mechanism by which cCMV increases the risk of ALL by encouraging proliferation of cells vulnerable to transformation.Collectively, these observations support the plausibility of CMV being involved in the cause of ALL.Aside from this plausibility, 2 independent studies have suggested an association between cCMV infection and ALL. 6,7e initial study by Francis et al 6 had a study design most similar to ours.The authors conducted a case-control study of newborn DBS from 268 ALL cases and 270 cancer-free controls.In comparison, our case-control study was nearly 10 times larger with a similarly strong, populationbased study design using newborn DBS to capture prenatal CMV exposure.The rates of CMV DNA positivity were different in our 2 studies, with the positive prevalence 7.5 times higher in the controls from Francis et al 6 compared with our controls.This raises the possibility of technical differences.
One major difference was the use by Francis et al 6 of droplet digital PCR to detect CMV DNA, while we used a quantitative PCR method optimized for detection of CMV DNA in newborn DBS. 11though droplet digital PCR has been shown to have increased precision over quantitative PCR in certain applications, the methods have similar sensitivity. 21,22However, contaminants that originate from the Guthrie card could influence the sensitivity of the quantitative PCR assay.Another possibility is the variation in quantity of starting material.Francis et al 6 reported using a quarter of a Guthrie spot, equivalent to about 33.2 mm 2 area, which was more than the punch area of 28.27 2 mm used in our study. 23Therefore, the likelihood of detecting cCMV could increase with the amount of material sampled.Although we cannot be certain what is driving the differences in CMV DNA prevalence at birth in the 2 studies, cCMV infection is detected in 0.45% of newborn DBS, which is consistent with our results. 11Furthermore, universal screening in high income countries have shown cCMV prevalence is consistently 0.6%. 24,25 outstanding question is whether the severity of cCMV is associated with future ALL risk.In Wiemels et al, 7 both congenital and early life acquired CMV infection was evaluated for future risk of hematologic malignant neoplasm in population-based registries of Sweden.Through passive screening, the prevalence of clinically recognized CMV infection at birth was 0.0066% in noncases and 0.088% in children who later received a diagnosis of hematologic malignant neoplasm (HR, 14.8; 95% CI, 4.8-45.9).Congenital CMV infection is clinically recognized in 10%-15% of all infected infants, as the majority of babies are asymptomatic. 26,27Therefore, the CMV infections investigated in Wiemels et al were likely severe. 28We do not know who in our study had a clinically recognized cCMV infection; however, we observed higher CMV levels in cases vs controls.Further investigation into this association is necessary.
Our findings suggest a CMV-ALL association may be specific to hyperdiploid ALL, consistent with recent reports from diagnostic specimens. 29

Strengths and Limitations
Our study had several strengths.A limitation of our study is this was a largely White population, which limited our ability to look in subgroups defined by demographics.Another limitation was Michigan DBS were stored at an ambient temperature, while DBS specimens collected after 2009 in the study by Francis et al 6 were at −20 °C.This could explain why we had a lower prevalence of CMV DNA, but ambient temperature has not been previously associated with lower quality 30 and our estimated prevalence was in line with other reports. 31Additionally, immunophenotype and cytogenetic subtype was available for a small portion of cases which contributed to imprecision of our point estimates.

Conclusions
This case-control study did not find evidence of an association between cCMV infection and pediatric ALL in our primary analysis.However, we detected an association among hyperdiploid ALL cases using small numbers.The partial support given here to the association of cCMV with ALL, previously seen in 2 epidemiologic studies, and its accumulating biologic plausibility suggest the need for continued research.Minnesota and Ontario jurisdictions have recently initiated universal cCMV screening, which may present locales to examine this emerging association further. 32,33Future work may also consider incorporating DNA methylation to help establish a mechanistic link between cCMV and ALL.Finally, work that also considers early childhood infections is needed to understand the association between this prevalent virus and ALL.

Table 1 .
Demographic Characteristics of ALL Cases and Controls

Table 1 .
Demographic Characteristics of ALL Cases and Controls (continued) a P values calculated by Pearson χ 2 statistic for categorical variables or by 2-sided t test for continuous variables.bIndicates matching factor.

Table 2 .
Prevalence of cCMV in Dried Blood Spots of ALL Cases Compared With Matched Controls, by Diagnostic Characteristics Abbreviations: ALL, acute lymphoblastic leukemia; cCMV, congenital cytomegalovirus; NA, not applicable; OR, odds ratio.aOddsratioand95%CIcalculatedby conditional logistic regression.bMultivariablemodeladjusted for mother's age at birth, maternal diabetes, birth weight, categorical gestational age, and presence of congenital anomalies: OR, 1.30 (95% CI, 0.52-3.24);oddsratiosand95%CIswere calculated first by conditional logistic regression among hyperdiploid ALL cases and their matched controls, but the model did not converge.cAnoddsratiocalculatedfor hyperdiploid cases compared with all controls (n = 4756) using exact methods: OR, 6.26 (95% CI, 1.44-27.19).dOddsratiocalculated in an unmatched analysis for hyperdiploid cases compared with all controls who had cases with available subtype data (2144) using exact methods: OR, 13.37 (95% CI, 1.25 -83.21).BControlsA, Viral load expressed as mean viral copies CMV/μg genomic DNA.t test P = .10.B, Viral load expressed as copies CMV/mL blood.t test P = .97.

Table 3 .
Congenital CMV Status Among ALL Cases and Controls, Stratified by Demographic Characteristics Abbreviations: ALL, acute lymphoblastic leukemia; CMV, cytomegalovirus; NA, not applicable; OR, odds ratio.a P values were calculated by Fisher exact test.
29togenetic subtype was available for 21% of cases.When stratified by ALL subtype, we found hyperdiploid ALL cases had 6.26 the odds of cCMV compared to all controls in our study, albeit based on only 2 exposed cases.Furthermore, hyperdiploid cases had 13.4 the odds of cCMV when compared with all controls who had cases with subtype data.High hyperdiploid ALL (generally defined as 50-67 chromosomes) was very recently shown to be associated with CMV by Gallant et al29in case-only analyses.Half of all bone marrow biopsies were CMV DNA positive, and bone marrow biopsies from B-cell ALL cases were more likely to be CMV positive compared with T-cell ALL (OR 1.63; 95% CI, 0.88-3.06).Considering just B-cell ALL, the biopsies from high hyperdiploid ALL cases were 1.7 times more likely to be CMV positive than ETV6-RUNX1 ALL, and 2.71 times more likely to be in the upper tertile of CMV-load (95% CI, 1.34-4.73).29Ourresults, in combination with those from Gallant et al, strongly suggest CMV is associated specifically to hyperdiploid ALL.
First, this study was a very large, population-based case-control study of 1189 ALL cases and 4756 controls, all with DBS obtained immediately after birth, and nearly 10 times larger than the prior study.Second, data linkage through the MBH enabled us to examine potential associations with birth characteristics and parental demographics and leukemia.Lastly, the use of nested samples taken shortly after birth make the temporality between measurement of cCMV and development of ALL clear.Since samples were collected immediately following birth, any detectable DNA would have been passed in utero.Furthermore, this is an advantage over the study by Wiemels et al, since they used medical records, which reported only clinically recognized CMV disease.