Page 267 - Haematologica Vol. 107 - September 2022

LETTER TO THE EDITOR
Cytomegalovirus proteins, maternal pregnancy cytokines, and their impact on neonatal immune cytokine profiles and acute lymphoblastic leukemogenesis in children
Early cytomegalovirus (CMV) infection and altered cyto- kine profiles at birth are associated with risk of childhood acute lymphoblastic leukemia (ALL).1-5 We examined neo- natal cytokine levels and CMV proteins in 130 children who contracted ALL later in life and 460 controls. We assessed the immunodominant viral coat protein (pp65) and CMV proteins that manipulate human immune function (CMV- IL-10, CMV-CXCL-1), which were detectable in most neo- natal samples and correlated with specific cytokine levels (IL-10, IL12, TGF-b1, and TNFa) CMV-IL-10 was positively associated with ALL risk. Neonatal cytokines, analyzed as a principal component loaded by IL-10, IL-12, and TNFa levels, were significantly different between cases and controls. Maternal mid-pregnancy cytokine expression was weakly correlated with cytokines at birth but did not differentiate childhood ALL cases and controls. In sum, the data provide preliminary indications that CMV viral ac- tivity during pregnancy may influence the neonatal cyto- kine profiles linked to risk of childhood ALL.
Maternal mid-pregnancy samples and matched neonatal blood spots from five California counties were obtained from the California Biobank.6 The study includes 137 cases born between November 1999 and 2009 and diagnosed with childhood ALL at the age of 0-14 years. Controls were frequency matched to cases on year of birth, sex, and race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, Asian/Pacific Islander, or other). Two 4.7-mm blood spot punches were treated to 160 mL of extraction buffer as described previously.1 Extracts were randomized to 96-well plates with each plate containing similar pro- portions of cases and controls and racial/ethnic groups. Twelve cytokines – IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, TNFa, VEGF, and IFN-γ, were measured using a Luminex bead–based assay (R&D Systems) incor- porating calibration standards, quality control samples, and blanks. Transforming growth factor (TGF-b1) and ar- ginase-2 were measured individually.7 Whole aqueous ma- ternal sera were analyzed for cytokines by Luminex.
CMV proteins were measured using customized Luminex assays. Three capture antibodies (CMV-IL-10: AF117, R&D systems, Minneapolis, MN;8 pp65: OAMA00562, Aviva sys- tems Biology Corp. San Diego, CA; UL146/vCXC1: AF620, R&D systems, Minneapolis, MN) were coupled to Luminex microspheres (Bio-rad kit). CMV protein standards (CMV-
IL-10:117-VL-025, R&D systems; pp65: CV001-1, Virusys Corp; UL146/vCXC1: 620-CM, R&D Systems) and blood spot extracts (10 uL/sample/test) were incubated with corresponding microspheres (5 uL/sample) at room tem- perature using a Curiox wash station, followed by 10 uL of 1:1,000 diluted biotinylated anti-CMV proteins antibodies (CMV-IL-10: BAF117, R&D systems; pp65: DPATB-H83463, Creative Diagnostics, New York, NY; UL146/vCXV1: BAF620, R&D Systems) and streptavidin-conjugated R-phycoeryth- rin 1:100 diluted stock (Bio-Rad Laboratories, Inc.). Lumi- nex measurements were converted to concentrations using a standard protein curve, and run in duplicate and averaged. For measurements that were below the level of detection, levels were assigned as one half the lowest level of detection.
Raw data were adjusted for batch, age of the blood spot, and the level of protein extracted. Variance Stabilizing Normalization (VSN)9 plus ComBat10 was used to prepro- cess and calibrate samples. Seven cases and 40 controls were excluded (due to quality control or technical failure), resulting in 130 cases and 460 controls for the final analy- sis. Pearson correlation coefficients were calculated among neonatal and maternal cytokines and CMV pro- teins. Multivariable logistic regression models were util- ized to assess associations between neonatal and maternal cytokines, CMV proteins, arginase-2 and risk of childhood ALL.
Most birth characteristics were not significantly different between cases and controls, apart from the frequency of cesarean section which was higher among cases (P=0.04; Online Supplementary Table S1), compatible with its status as a known risk factor for ALL.11 Cytokines exhibited ex- tensive correlation in children and with CMV proteins (Fig- ure 1). CMV-IL-10 was inversely correlated with human IL-10, as well as IL-12p70, TNF-a, VEGF, arginase-2, and weakly inversely correlated with the other CMV-derived cytokine CMV-CXC-1. CMV-pp65, the coat protein of the CMV virus itself, exhibited similar correlations as CMV-IL- 10. CMV-CXC-1 demonstrated significant inverse associ- ation with TGF-b1 which was not observed for the other CMV proteins (Figure 1).
While some correlations between neonatal cytokines ap- proached rho=0.6 (Figure 1), all correlations between ma- ternal cytokines in mid-pregnancy and child cytokines at
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