Project Grant R21ES036341

LEVERAGING MUTATIONAL ANALYSIS OF CELL-FREE DNA TO IDENTIFY CARCINOGENIC EXPOSURE AND ENABLE EARLY DETECTION OF CANCER - PROJECT SUMMARY / ABSTRACT PRELIMINARY WORK HAS SHOWN THAT TWO DIFFERENT ENVIRONMENTAL HEPATOCARCINOGENS, AFLATOXIN B1 (AFB1) AND N-NITROSODIMETHYLAMINE (NDMA), PRODUCE HIGH-RESOLUTION MUTATIONAL SPECTRA (HRMS) THAT OCCUR SHORTLY AFTER CARCINOGEN EXPOSURE, ARE DISTINCT FROM ONE ANOTHER, AND ARE MECHANISTICALLY IN ACCORD WITH THE ESTABLISHED MUTATIONAL PROPERTIES OF THE DNA ADDUCTS THESE AGENTS FORM. EXTENDING THAT WORK, THE GOAL OF THE PROPOSED PROJECT IS TO DEVELOP A "BLOOD-BASED" ANALYTICAL TOOL ENABLING RAPID DETECTION OF THE MUTATIONAL PROFILES OF THESE AGENTS. THE HYPOTHESIS TO BE TESTED IS THAT MECHANISTICALLY INFORMATIVE MUTATIONAL FINGERPRINTS OF ENVIRONMENTAL TOXICANTS ARE PRESENT IN CIRCULATING CELL-FREE DNA (CFDNA) OBTAINED FROM BLOOD OF EXPOSED INDIVIDUALS. THIS PROJECT WILL ADDRESS A GAP IN KNOWLEDGE CONNECTING ENVIRONMENTAL EXPOSURES TO CANCER RISK. SEQUENCING OF CANCER GENOMES HAS REVEALED ~100 MUTATIONAL PATTERNS TERMED "SIGNATURES," WITH SOME SIGNATURES SHOWING SIMILARITY TO MUTATIONAL SPECTRA PRODUCED BY KNOWN ENVIRONMENTAL CARCINOGENS (E.G., UV LIGHT, AFB1, BENZO(A)PYRENE). CURRENTLY, THERE IS NO FACILE WAY TO MEASURE THE GENETIC CONSEQUENCES OF PRIOR GENOTOXIC EXPOSURES BECAUSE OF: (1) THE INVASIVE PROCEDURES REQUIRED TO OBTAIN TISSUE SAMPLES, AND (2) THE INSENSITIVITY OF "TYPICAL" DNA SEQUENCING METHODS. THIS PROPOSAL WILL TAKE ADVANTAGE OF RECENT ADVANCES TO OVERCOME THESE LIMITATIONS. CONSISTING OF FRAGMENTS ~170 BP LONG, CFDNA ORIGINATES FROM NORMAL CELL TURNOVER AS WELL AS FROM APOPTOTIC AND NECROTIC CELLS FOLLOWING EXPOSURE TO TOXINS. CONVENTIONAL NEXTGEN DNA SEQUENCING TOOLS CANNOT RELIABLY IDENTIFY THE LOW LEVELS OF MUTATIONS THAT ARE PRESENT IN CFDNA. TO OVERCOME THIS SENSITIVITY LIMITATION, WE SHALL USE DUPLEX CONSENSUS SEQUENCING, WHICH WE HAVE SHOWN AFFORDS UP TO 104-FOLD HIGHER ACCURACY/SENSITIVITY OVER CONVENTIONAL SEQUENCING. THE SPECIFIC AIMS WILL DETERMINE IF HRMS PREVIOUSLY OBSERVED IN MOUSE LIVER GENOMIC DNA FOLLOWING TREATMENT WITH AFB1 AND NDMA CAN BE IDENTIFIED IN CFDNA IN BLOOD. AIM 1 WILL QUANTIFY THE LEVELS OF CFDNA IN BLOOD AFTER TREATMENT. CIRCULATING CFDNA WILL BE ASSAYED AT SEVERAL TIMES AFTER TREATMENT TO DETERMINE THE TEMPORAL RELATIONSHIP BETWEEN EXPOSURE, HEPATOTOXICITY AND THE LEVELS OF CFDNA IN BLOOD. AIM 2 WILL DETERMINE COMPOUND-SPECIFIC MUTATIONAL SPECTRA IN CFDNA AND COMPARE THEM TO SPECTRA OBTAINED FROM GENOMIC DNA FROM TARGET TISSUE (LIVER) AS WELL AS WHITE BLOOD CELLS FROM BLOOD, LYMPH NODES AND THYMUS. AIM 3 WILL EXAMINE MUTATIONAL SPECTRA IN LIVER-SPECIFIC CFDNA GENERATED BY CHALLENGING THE ANIMALS WITH MILD HEPATOTOXICANTS (E.G., ETHANOL, ACETAMINOPHEN), WHICH TRIGGER INCREASED CELL TURNOVER. THE SUCCESS OF THESE STUDIES WILL PROVIDE AN INNOVATIVE APPROACH TO MUTATIONAL FINGERPRINTING OF ENVIRONMENTAL EXPOSURES THAT COULD, IN TURN, LEAD TO PREDICTIVE BIOMARKERS THAT WOULD TRIGGER INTERVENTIONS TO LIMIT FUTURE EXPOSURES AND REDUCE HEALTHCARE NEEDS BY PREVENTING THE DEVELOPMENT OF ADVANCED DISEASE.