Untargeted Adductomics of Newborn Dried Blood Spots to Identify Exposures Associated with Childhood Leukemia

The causes of cancer are likely to involve a combination of genetic factors, exposures, and random chance. However, current evidence suggests between a third and a half of cancers may be preventable, and that exposures may play an important role in the etiology of cancer. In fact, it is possible that the most common type of cancer among children, childhood leukemia, may be a preventable disease. Current scientific knowledge suggests early life exposures, including exposures occurring in utero, play critical roles in the development of childhood leukemia. However, few risk factors have been established, and the underlying disease mechanisms remain elusive./nThe overarching goal of this dissertation research was to address this gap in knowledge by performing an untargeted adductomics analysis of archived newborn dried blood spots (DBS) to identify risk factors for childhood leukemia resulting from in utero exposures. Metabolism of chemicals derived from the diet, exposures to xenobiotics, the microbiome, and lifestyle factors (e.g., smoking, alcohol consumption) produce electrophiles that react with nucleophilic sites in circulating proteins, notably Cys34 of human serum albumin (HSA). Since HSA has a residence time of 28 days, HSA-Cys34 adducts measured in archived newborn DBS reflect systemic exposures during the last month of gestation and allow us to explore potential risk factors resulting from in utero exposures./nHere, we have developed an untargeted adductomics method to detect HSA-Cys34 adducts in 4.7-mm punches from DBS (equivalent to 5 – 8 μL of whole blood). The workflow includes extraction of proteins from DBS, measurement of hemoglobin to normalize for blood volume, addition of methanol to enrich HSA by precipitation of hemoglobin and other interfering proteins, digestion with trypsin, and detection of HSA-Cys34 adducts via nanoflow liquid chromatography-high resolution mass spectrometry. As proof-of-concept, we tested the DBS-adductomics method with 49 archived DBS collected from newborns whose mothers either actively smoked or were nonsmokers during pregnancy. A novel normalization method (‘scone’) was used to remove unwanted technical variation arising from: HSA digestion, blood volume, DBS age, mass spectrometry analysis, and batch effects. A total of 26 HSA-Cys34 adducts were detected, including Cys34 oxidation products, mixed disulfides with low-molecular-weight thiols (e.g., cysteine, homocysteine, glutathione, cysteinylglycine, etc.), and other modifications. Using an ensemble of variable selection methods, including both linear and nonlinear models, we found that the Cys34 adduct of cyanide consistently discriminated between newborns of smoking and nonsmoking mothers with a fold change (smoking/nonsmoking) of 1.31 and a cross-validated area under the estimated receiver operating characteristic curve (cvAUC) of 0.79. Indeed, hydrogen cyanide is a component of cigarette smoke, and these results indicated that DBS-based adductomics is suitable for investigating in utero exposures to reactive electrophiles that may influence disease risks later in life./nWe then applied the DBS-adductomics method to analyze 783 archived newborn DBS collected from childhood leukemia cases and matched controls participating in the California Childhood Leukemia Study. Childhood leukemia cases included the two main subtypes, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), with additional molecular subtypes for ALL. After data preprocessing and normalization, a combination of linear and nonlinear models were used to identify adducts that discriminated between leukemia cases and controls. Of the 28 Cys34 adducts that were detected, none was predictive of ALL overall. However, several adducts showed increases in adduct abundances in subgroups of cases with T-cell ALL and B-cell ALL with t(12;21) translocations. For both subgroups, elevated levels of adducts of reactive carbonyl species in cases may suggest that oxidative stress and lipid peroxidation had influenced adduct production. Regarding AML, the Cys34 homocysteine adduct (with loss of H2O) was found to consistently discriminate between AML cases and controls with a fold change (case/control) of 0.66. Since homocysteine is an important intermediate in the folate-mediated one-carbon metabolism, this indicates alterations in epigenetic regulations and folate status may be involved in the etiology of AML. Moreover, because lower homocysteine levels were detected in newborn DBS collected years before AML cases were diagnosed, biological changes involved in the initiation of AML may be present at birth and that there may be avenues for preventing the disease. Since this was a hypothesis-generating study, these findings warrant replication in follow-up studies with larger sample sizes of the various subtypes. Future integrated analyses with other omics (e.g., genomics, metabolomics, epigenomics) will be key to obtaining a full picture of the disease mechanisms regarding childhood leukemia.

Advisor: Rappaport, Stephen M