>> WELCOME TO THE MAY 15th 2018 DNA REPAIR INTEREST GROUP VIDEO CONFERENCE. WE HAVE ONE MORE IN JUNE. DR. LUDMILL ALEXANDROFF FROM UC SAN DIEGO WILL BE COMING HERE AND SPEAKING ABOUT A COMPREHENSIVE MULTIPLE SIGNATURE IN THE HUMAN CANCER. WE'LL BE HAVING A SUMMER BREAK IN JULY AND AUGUST. AND WE'LL START AGAIN IN SEPTEMBER. THOSE OF YOU WHO ARE HEADS OF THE DIFFERENT SITES CAN THEN NOMINATE SPEAKERS FOR NEXT SEASON. JUST ACCEPTED E-MAILS TO WILL OR TO ME TO DO THAT. SO WE'RE PLEASED TO HAVE TODAY'S SPEAKER FROM SUNI, DR. TOM ROSEN QUIOF THE WILL SPEAK ABOUT REPAIR RESISTANT ARISTOLOCHIC DERIVATIVE ADDUCTS AND MAKE SURE EVERYBODY ELSE IS MUTED EXCEPT FOR AT STONY BROOK. OKAY. SO OUR SPEAKER TODAY IS THOM ROSENQUIST, ASSOCIATE PROFESSOR OF PHARMACOLOGICAL SCIENCES AT STONY BROOK. HE RECEIVED HIS Ph.D. FROM THE UNIVERSITY OF WISCONSIN AND CONDUCTED POST DOCTORAL STUDIES WITH GAIL MARTIN AND DEVELOPMENTAL BIOLOGY AT UCSF. HE JOINED OUR FACULTY IN 1995 SETTING UP AN INDEPENDENT RESEARCH PROGRAM WHILE ESTABLISHING AT THE SAME TIME AND THEN DIRECTING A MOGGOR MOUSE GENOMICS FACILITY. SOON AFTER HIS ARRIVAL TOM BECAME INVOLVED IN DNA REPAIR THROUGH HIS DISCOVERY AND CHARACTERIZATION OF MOUSE AGUAN, AND LATER TRANSITIONING FROM MOUSE TO HUMAN RESEARCH, HE CO-DISCOVERED THENY O 1 FAMILY OF DNA GLUE MARIOUS COSLACE. --GLUE MARIOUS COSLACE, HE THEN EXTENDED HIS STUDIES TO MUTE O GENESIS AND HE WILL SPEAK TO US TODAY ON THE SUBJECT REPAIR-RESISTANT STONY BROOK STOL ACID DNA ADDUCTS. TOM? >> THANK YOU ARTHUR FOR THAT INTRODUCTION, AS MOST YOU YOU ARE AWARE, ARTHUR GROMAN HERE AT STONY BROOK HAS A LARGE GROUP OF RESEARCHERS THAT ARE INDEPENDENT WITH HIM AND UNDER HIS DIRECTION HAS BEEN INTERESTED IN THE ARISTOLOCHIC ACID FIELD. THIS IS A NEVER EXPANDING FIELD. EVERY YEAR IT SEEMS LIKE THERE'S ANOTHER DISEASE THAT ARISTOLOCHIC IS--[NO AUDIO ]--SIGNATURES TO DERIVE DIRECTLY FROM THE ARISTOLOCHIC ACID AND IN THE TITLE THE UNIQUE DNA ADDUCTS AND THE TOOL FOR DETECTING THOSE AND HOW THEY CAN BE APPLIED TO NOT ONLY MOLECULAR EPIDEMIOLOGY BUT ALSO IMPLY THAT THERE MAY BE--THESE ADDUCTS MAY BE AN ONGOING HEALTH FOR THE HARBOR THESE ADDUCTS.E WHO- NOW ARISTOLOCHIC IS A GEANUS OF PLANTS THAT GROW THROUGHOUT THE WORLD AND TEMPERATE AND TROPICAL REGIONS. WHEREVER PEOPLE ARE THEY USED THIS SPECIES TO GENERATE HERBAL REMEDIES AND TRADITIONAL REMEDIES FOR A VARIETY OF MALADIES AND I'VE SHOWN HERE 1 SUCH SPECIES OF ARISTOLOCHIC DERMATITIS THIS IS ALSO KNOWN AS BIRTH WART. IT'S USED IN EUROPE FOR COMPLICATIONS IN CHILD BIRTH. THE MOLECULE SHOWNOT RIGHT IS STOL TOL ACID. IT'S A FAMILY OF NIGHT ROUGH ATOM PHINANTHRINE, CARBOLIC ACIDS. THIS PARTICULAR 1 IS ACID 1 WHICH IS A MAJOR SPECIES WHICH WHICH CAUSES MOST OF THE HUMAN HEALTH PROBLEMS. NOW AS I IMPLIED THROUGHOUT HISTORY ARISTOLOCHIC HAS BEEN USED IN HERBAL REMETABOLISM REMETABOLISM--REMEDIES, YOU SEE LITERATURE FROM ARISTOLO CHIA, AND THIS IS A REMEDY FOR EDEMA, USING ARISTOLOCHIC. NOW AT 1 POINT WHICH ARTHUR AS MADE OVER THE YEARS WHICH IS REALLY REMARKABLE IS OVER 2500 YEARS NO TOXICITIES WERE REPORTED WITH THE USE OF ARISTOLOCHICKIA. THAT IS UNTIL LAST SEVERAL DECADES WHEN ATTEMPTS WERE MADE TO TURN ARISTOLOCHIC PRODUCTS INTO MEDICINES. IN THE LATE EARLY 1960S, CHEMO THERAPIES WERE PROPOSED BASED ON ARISTOLOCHIC BUT AS SOON AS THEY HAD CLINICAL TRIALS, WE REALIZED THIS WAS A VERY POTENT NEFF ROW TOCKS AND I KNOW CAUSED RENAL FAILURE IN THE RECIPIENTS. SO IT'S USE IN MEDICAL APPROACHES IT WAS DROPPED THOUGH IT IS SORT OF AN INTERESTING RESEARCH TOOL IT'S A FOSTER NURSED FOCUSED ON LIAISON PHASE LITERATURE, IT'S BEEN USED IN THAT LITERATURE OVER THE DECADES. IT WAS SHOWN TO BE A MUTA GEN AND PROHIBITED FOR FURTHER USE AND DEVELOPMENT OF MEDICINE'S HERE IN THE UNITED STATES AND THROUGHOUT THE WEST. HOWEVER BECAUSE IT WAS A RESEARCH TOOL, SEVERAL USEFUL TOOLS FOR OUR PURPOSES OR IN A STUDY THAT SET UP THE GROUND WORK FOR OUR STUDIES WORK DONE OVER THE DECADES NOW AND I WILL REFER TO THOSE THROUGHOUT THE TALK. NOW OUR MODERN APPRECIATION THAT THIS IS PROBABLY HAS SOME COMPONENT OF PUBLIC HEALTH CONCERN CAME FROM SORT OF AN UNNATURAL HUMAN EXPERIMENT WHICH HAPPENED IN BRUSSELS IN BELGIUM IN THE EARLY 90S. SO ABOUT 2000 WOMEN IN THE SPACE OF A FEW YEARS SHOWED UP AT HEALTH CLINICS WITH RENAL FAILURE. NOW THIS IS REALLY UNUSUAL FOR WOMEN WHO ARE IN THEIR MIDDLE AGES TO HAVE RENAL FAILURE. SO THIS TYPE OF CLUSTER WAS REALLY DREW THE ATTENTION OF THE NEFFROLOGYST IN THE BRUSSELS AREA. AND SEVERAL OF THESE WOMEN, ABOUT 5%--A FAIR MARGIN, ABOUT 5% HAD TO HAVE THEIR KID MES AREY MOVED AND HAD TO URPD GO DIALYSIS. WHEN THEIR KIDNEYS WERE REMOVED AND EXAMINED, ANOTHER SURPRISE AT ABOUT HALF OF THEM WERE--HAD DISPLACE UROTHELIUM IN THE PELVIS AND UREATER, NOW THIS IS THE UPPER AREA TRACT CANCER IS VERY RARE, THERE'S ABOUT 5% OF THE CANCERS MAINLY MOST OF THEM ARE IN THE LOWER TRACT IN THE BLADDER. SO I HAD THIS TYPE OF CLUSTER, 2 TYPES OF REALLY UNUSUAL CLUSTERS ASSOCIATED WITH THIS TOGETHER, REALLY DROW A LOT OF ATTENTION. AFTER A LOT OF DETECTIVE WORK IT WAS REALIZED THAT ALL MEAS WOMEN COULD BE CHECKED BACK TO ATTEND A PARTICLAR WEIGHT LOSS CLINIC IN BRUSSELS WHICH USED THE MIXTURE OF DRUGS, PLANT EXTRACTS TO INDUCE WEIGHT LOSS. NOW OVER THE COURSE OF 13 MONTHS IT WAS DETERMINED THIS WEIGHT LOSS CLINIC HAD DUE TO A TRANSLATION ERROR HAD INADVERTENTLY SUBSTITUTED AN ARISTOLOCHIC PRODUCT OR A STEPHANIA PRODUCT AND THEY WERE TREATED WITH DOSES THAT WOULD NORMALLY NEVER BE SEEN AND UNDER A SHORT TIME UNDERWENT RENAL FAILURE AND INITIATION OF ARISTOLOCHIC CANCER NOW THERE'S UNCREDIBLE HIGH RATE OF CANCER LED TO THE RECOMMENDATION AT ANY TIME THESE ARISTOLOCHIC RENAL FAILURES INVOLVED TO JUST REMOVE THE KIDNEYS, REMOVE THE URETER AND DRASTIC CLINICAL OUTCOME. NOW THIS ASSOCIATION WITH THE ARISTOLOCHIC WAS DRICH HOME WHEN LOOKING AT THE SURGICAL SAMPLES THAT WERE REMOVED FROM THESE PATIENTS AND EXTRACTING DNA, WHO HAD BEEN SORT OF WORKING ON AT THE BENCH AND THE ACID BUT HAD DEVELOPED P32 POST LABELING& METHODS TO DETECT THE ADDICTS, HE WAS BROUGHT IN AND HE FOUND DMA ADDUCTS IN EVERY SINGLE PEASHT WAS THAT INVOLVED IN THIS COHORT. SO IT CAME OUT OF THIS THAT APPRECIATION AT ARISTOLOCHIC CAN CAUSE 2 DISEASES. BOTH IN THIS CASE, BOTH IN THE KIDNEY. NEPHROPATHY DUE TO WASTING OF RENAL TUBULES AND THE RENAL CORTEX, SOME NEUROECTODERMALCROSEIS OF CELLS IN THE TUBE ALLS AND ATROPHY OF THE TUBULES AND FIBROSIS FILLING IN THE SPACE AND LOSS OF FUNCTION AND LEADING TO END STAGE RENAL DISEASE. IN THE NEARBY TISSUE OF THE RENAL PELVIS, THE UROLOGYST O THELIUM, CONTINUOUS WITH THE URETER, YOU ALSO GET CANCER SO SOMETIMES THESE CANCERS ARE INSIDE THE KIDNEY, SOMETIMES THEY'RE IN THE URETER, WHERE THIS NODULE IS A TUMOR THIS URETER. SO THIS HISTOLOGICAL SLIDE SHOWS HOW DRAMATIC THIS PATTERN OF WASTING IN THE UTTER CORTEX IS, AND ON THE LEFT HERE THIS IS WHAT'S LEFT OF A KIDNEY WHEN THE ENTIRE OUTER REGION OF THE KIDNEY HAS DIED OFF JUST A TINY REMNANT AND YOU CAN SEE THIS PATTERN IN THIS OUTSIDE OF THE KIDNEY, THIS GOES FROM--I DIDN'T PUT ANAXIS ON HERE, THIS IS THE OUTER CORTEX ANDINNER PORTION OF THE KIDNEY, ALL OF THE TUBULES ARE GONE FROM THE OUTER CORTEX OF THIS KIDNEY AND THE BLUE STAINING SHOWS EXTENSIVE FIBROSIS. ANOTHER FEATURE THAT'S SORT OF STRANGE IS THE GLUE MARIOLE I ARE NOT EFFECTED SO WHATEVER IS ACTIVE IS ACTIVE IN THE TUBUALS OF THE KIDNEY BUT IN THE GLOMMURULE I, WE HAVE A RARE SPECTRUM OF HISTOLOGICAL FEATURES. HOWEVER IT'S NOT--WASN'T UNIQUE. BY HAPPEN CHANCE 1 OF THE PATHOLOGISTS WORKING ON THESE CASES, DR. KAZANZ, HAD SEEN THIS NOW THE CONTEXT HE HAD SEEN IT WAS UNEXPLAINED NEPHROPATHY FROM A DIFFERENT PART OF EUROPE. THIS IS--WAS KNOWN AT THE TIME AS BALKAN--KANA ENDEMIC NEUROPATHY IT EFFECTS RURAL FARMING FAMILIES ALONG THE DANIEL RIVER AND ORIGINALLY FOUND IN YUGOSLAVIA AND IT'S ONGOING IN CROATIA, BOSNIA, AND OTHER STATES ARE ROMANNIA AND BULGARRIA. NOW WILL THIS BE PRESENT IN 1 FARMING VIMMAGE, AN ADJACENT FARMING VILLAGE IT WON'T BE SO THE ENDEMIC VILLAGES ARE WHEAT FARMERS, THEY FARM THEIR OWN WHEAT. THEY ARE LOCATED IN THE LOWLANDS WHICH BECOME FLOODED FREQUENTLY. THE PATTERN IS THAT GIVEN--IT'LL BE LOCALIZED WITHIN A HOUSEHOLDS, WHERE SEVERAL MEMBERS OF THE SAME HOUSEHOLD WILL--AT THE END OF THEIR 50S AND 60S WILL HAVE RENAL DISEASE OR DEVELOP THE ASSOCIATED UPPER TRACK CANCER. IT TAKES A LONG TIME TO DEVELOP. THIS IS THE CONTRACT WITH THE BELGIAN SITUATION. THIS TAKES AT LEAST 15 YEARS TO DEVELOP. PEOPLE HAVE TO LIVE IN THESE VELLAGES FOR 15 YEARS BEFORE THEY START TO HAVE SYMPTOMS. CHILDREN WHO MOVE AWAY BEFORE THEY'RE ADULTS DO NOT DEVELOP THE SYMPTOMS. HOWEVER THE HISTOLOGY IS THE SAME AS WHAT WAS SEEN IN THE BULGEIUM WOMEN AND THE UPPER URINARY TRACT CANCER IS HIGHLY PREVALENT. AND WE SHOW THAT THE ADDUCTS WERE PRESENT IN SAMPLES IN THESE PATIENTS AS WELL. NOW THE ROOT OF EXPOSURE IN THIS CASE, WE THINK IS DUE TO DIETARY CONTAMINATION, THESE PEOPLE FARM THEIR OWN WHEAT, THEY USE METHODS THAT THEY USED FOR A LONG TIME AND IF YOU GO AND JUST TAKE PICTURES AND WALK AROUND THE PICTURES AND YOU CAN SEE THE ARISTOLOCHIC GROWING IN THE FIELDS. THESE ARE THE SEEDS THAT MATURE AT THE SAME TIME AND GET HARVESTED ALONG WITH THE WHEAT AND TEND TO USE LOCAL MILLS THAT HAVEN'T CHANGED OVER THE DECADES, THE TECHNIQUES FOR CYSTING OUT THE SEEDS ARE NOT THAT REFINED AND THEY PRODUCE FLOWER FROM THEIR OWN HARVEST, THEY TAKE HALF THE FLOWER BACK TO THEIR OWN HOUSE AND HALF GOES TO MARKET. AND THEN DURING THE YEAR THEY MAKE THEIR OWN BRED AND THROUGHOUT THE ENTIRE YEAR THEY'RE CO CONTAMINATED WITH THE REST OF THE LO KIA SEEDS. WE TRIED TO DETECT THOSE AND THE DPLIEWTEN MAKE ITS ALMOST IMPOSSIBLE, WE VBT DEVELOPED METHODS YET TO DO THAT HOWEVER YOU CAN EXTRACT DNA FROM FLOWER AND SO WE'VE DONE THAT AND THERE'S QUANTITATIVE PC R FOR THOSE SEQUENCES AND ALMOST HALF OF THOSE FLOWER FAMILIES WE CAN READILY DETECT THE DNA SO WE THINK THIS IS PROBABLY THE ROOT OF EXPOSURE HOWEVER I SHOULD MENTION THERE WAS ANOTHER HYPOTHESIS WHICH IS THAT WHEN THE FIELDS ARE PLOWED UNDER THE PLANTS ARE PLOWED UNDER AND THE ACID WILL BE RELEASED IN THE SOIL AND TAKEN UP BY SUBSEQUENT PLANTINGS. THIS HAS BEEN SHOWN TO WORK IN THE GROWN HOUSE, WHETHER IT WORKS IN THE FIELD OR NOT IS A QUESTION FOR DEBATE BUT IT'S AN IDEA THAT'S OUT THERE. THERE'S A COUPLE CASES IN EUROPE. I MENTIONED THAT THE ARISTOLOCHIC IS A COMPONENT OF MEDICINE THROUGHOUT THE WORLD AND TODAY IT'S USED EXTENSIVELY IN CHIENIZE MEDICINE AND EFFECTS INDIA THROUGHOUT SOUTHEAST ASIA, TAIWAN IN PARTICULAR IS A PLACE THAT HAS THE HIGHEST RATE OF THIS RARE CANCER IN THE WORLD, ABOUT 4 PER HUNDRED THOUSAND. THIS IS ABOUT 4 TIMES WHAT YOU WOULD SEE HERE IN THE UNITED STATES. IT'S ROUGHLY, YOU KNOW PUT INTO PERSPECTIVE, ROUGHLY THE RATE OF ESOPHAGEAL CANCER HERE IN THE UNITED STATES. FOR A TIME PERIOD THE NATIONAL HEALTH SERVICE IN TAIWAN RE'EM BUSKERRED FOR PEOPLE USING PRESCRIPTIONS WITH ARISTOLOCHIC, AND DURING A 6 YEAR PERIOD, FOUND THAT 1 IN 3 ADULTS CONTAINED A PRESCRIPTION TO ARISTOLOCHIC. THE EXPOSURE IS NEAR UBIQUITOUS IN TAIWAN. IN 2003 STAY STOPPED THAT PRACTICE HOWEVER, THERE'S THE TRADITIONAL PRACTICES ARE STILL GOING ON. NOW 8 MILLION PEOPLE IN TAIWAN, WE KNOW HAVE TAKEN ARISTOLOCHIC KIA AND THEY'RE LIKE, THIS TRANSLATES TO NEIGHBORING CHINA TO ABOUT A HUNDRED MILLION PEOPLE. SO THIS IS A VERY LARGE PROBLEM IN ASIA AND SOUTHEAST ASIA AND EAST ASIA, HOWEVER THERE ARE SPORADIC CASES HERE IN THE UNITED STATES, AND EVEN THOUGH WE'VE SORT OF BANNED ITS USAGE YOU CAN JUST GO ON THE INTERNET, BEFORE EVERY TALK, I UPDATE THIS. THIS IS JUST FROM SUNDAY GOING THROUGH AMAZON.COM, 2 PRODUCTS BEING SOLD FOR MEDICINAL PURPOSES SPECIFICALLY THAT YOU COULD BUY. IN ADDITION THERE'S DOZENS OF PRODUCTS WHICH ARE THE DRIED SEEDS OR LEAVES, ROOTS OF THE PLANTS WHICH YOU CAN MAKE YOUR OWN REMEDIES FROM. SO JUST TO SUMMARIZE THEN, WHAT STARTED AS SORT OF AN UNFORTUNATE INCIDENT OF, YOU KNOW A TRANSLATION ERROR IN BELGIUM, AS LED TO THE KEY SOLUTION TO AN ENDEMIC MEDICAL MYSTERY IN EUROPE AND THIS IS SORT OF ONGOING STILL TO THIS DAY. AND ALSO, GAVE US THE APPRECIATION THAT THIS IS REALLY A GLOBAL HEALTH RISK OF VERY SIGNIFICANT PROPORTIONS IN EAST ASIA. SO I WANT TO SWITCH NOW AND TALK MORE ABOUT THE MOLECULAR EPIDEMIOLOGY TOOLS, THE TYPE OF BIOMARKERS WE CAN USE NOW THAT WE KNOW THIS IS A LARGE PROBLEM, WE WOULD LIKE TO PUT--GET A FEEL FOR WHAT REALLY THE SCOPE IS OF BOTH THE GLOBAL SCOPE AND ALSO THE CONSEQUENCE FOR INDIVIDUALS WHO HAVE BEEN EXPOSED. SO BIOMARERS I'LL TALK ABOUT FIRST IS, THESE DECEASES ARE MAINLY CANCERS, CANCERS ARE BASED GENETICALLY ON MUTATIONS, WE WOULD LIKE TO UNDERSTAND THAT TUMOR MUTATIONS--NOW HISTOLOGICALLY THE CANCER CAUSED BY THE ACID LOOKS LIKE CANCER CAUSED BY ANY OTHER AGENT. HOWEVER ONCE YOU LOOK AT THE GENOME, YOU START TO DISCERN, YOU KNOW WHAT MAY HAVE CAUSED THESE TUMORS AND THE 1S THAT ARE CAUSED BY ARISTOLOCHIC ARE QUITE UNIQUE. THESE COME IN 2 SUBSPECIES. FIRST THE DRIVER MUTATIONS IS WHAT WE NORMALLY THINK OF, WE THINK ABOUT TUMOR SUPPRESSOR GENES, WE THINK ABOUT ONCOGENES AND WE WOULD LIKE TO KNOW WHAT THOSE MUTATIONS ARE AND IF THOSE MUTATIONS FIT THE PATTERN, THAT'S CORRELATED WITH THE SPECIFIC AGENT, WE CAN THEN ARGUE THAT THAT AGENT CAUSED THAT CANCER. NOW MORE--WIDE SPREAD IS THE GENOME WIDE MUTATION SIGNATURE, THIS DOESN'T ARGUE THE CAUSE OF NATURE OF AN AGENT BUT WILL ARGUE THAT IT'S EXPOSED TO THE AGENT. SO THIS IS A BIOMARKER OF EXPOSURE, NOW THE ADVANT KNOWLEDGE OF THIS IS SEQUENCING 1 OR 2 TUMORS CAN YOU DETERMINE A MUTATIONAL SIGNATURE, YOU HAVE TO SEQUENCE HUNDREDS OF, DOZENS OR HUNDREDS OF DRIVER MUTATIONS FROM DIFFERENT TUMORS IN ORDER TO ESTABLISH A PATTERN OF DRIVER MUTATION OCCURRENCE. NOW I'LL TALK ABOUT WHAT WAS IN THE TITLE OF THE TALK, A. L. L. DERIVED DNA DRIVER ADDUCTS WHICH CAN START TO PAINT A PICTURE OF NOT JUST EXPOSURE BUT ALSO WHETHER OR NOT THE AGENTS COULD CAUSE THE CANCER AS WELL. SO FIRST I'LL LOOK AT DRIVER GENES AND IF YOU ARE STUDYING UPPER TRACT URINARY CARCINOMAS YOU THINK OF P53 SINCE ABOUT HALF OF THEM HAVE MUTATIONS IN TP53. THIS SUMMARIZES A LOT OF WORK DONE BY OUR GROUP AND ALSO CORROBORATED BY OTHER GROUPS. MAKI, MARIA, WORKING WITH DR. GROMAN AND OUR COLLABORATORS IN TAIWAN. DR. CHEN AT NATIONAL TAIWAN UNIVERSITY AND COLLABORATORS IN CROATIA, [INDISCERNIBLE] AT UNIVERSITY OF [INDISCERNIBLE] PROVIDED A LOT OF SAMPLES. IF WE LOOK AT WORLD WIDE, THE DATABASES OF SEQUENCES OF THE TP53 AND BLADDER CANCERS OR SPECIFICALLY UPPER TRACK CANCERS, YOU FIND THAT THEY'RE PREDOMAINIDATED BY CDT MUTATIONS. HOWEVER, THE ARISTOLOCHIC ACID TP53 GENES JUMP RIGHT OUT AS BEING VERY DIFFERENT. THEY'RE PREDOMINATED BY ADT MUTATIONS NOT JUST IN THE TP53 GENE BUT IN CANCERS IN GENERAL. ABOUT ONLY 5-10% OF MUTATIONS IN CANCERS AND IN HUMANS ARE A-T MUTATIONS SO THIS IS A DRAMATIC SIGN. NOW A LOT OF WORK IN TISSUE CULTURE SYSTEMS, MY VARIOU LABS SHOWED THIS IS ACTUALLY, IF YOU TREAT THEM WITH ARISTOLOCHIC ACID YOU GET A-T TRANSFERS TRANSFERS AND IT'S NOT A REAL SURPRISE BUT THIS TYPE OF DRAMATIC EFFECT REALLY IS PRETTY STRIKING. NOW IF YOU LOOK INSIDE THE TP53 AND ASK WHERE ARE THESE TRANSVERSIONS ARE LOCATED, THIS IS EVEN MORE STRIKING. THEY'RE ALMOST ALWAYSOT NONTRANSCRIBED STRAND. VERY RARELY FOUNDOT TRANSCRIBED STAND EMPLOY SO THIS IS A HALLMARK OF TRANSCRIPTION COUPLED REPAIR, THE TRANSCRIBED STRAND IS SURVEILLED BY RNA POLYMER ACE, THE NONTRANSCRIBED STRAND ISN'T. FURTHER MORE THERE ARE HOT SPOTS FOR THESE MUTATIONS. QUITE OFTEN THOAR FOUND OF JURCHGZS OF INTRONSTRONS AND AXONS AT THE SPLICE RECEPTOR SITES. THE RED ARE SAMPLES THAT ARE FOUND THAT WERE OBTAINED FROM TAIWAN, THE BLACK FROM THE BALKANs AND CROATIA, SO WHATEVER AGENT CAUSED THE TAIWANESE DISEASE AND PROBABLY CALLED THE BALKANESE DISEASE, AND CAUSED THE ARISTOLOCHIC ACID. SO THIS IS DRIVER MUTATIONS MUTATIONS AND IN AND OF ITS IS A SMOKING GUN, WE ALSO EXTENDED THIS GENOME WIDE TO LOOK AT THE SIGNATURE. ONE OF THE FIRST THINGS THAT CAME OUT OF SEQUENCING THESE WHOLE EXOMES FROM TUMORS FROM TAIWAN. THE 1S THAT WERE ASSOCIATED WITH THE ARISTOLOCHIC ACID HAVE A LOT OF MUTATIONS. THIS DIAGRAM IS REVIEWED BY FOGEL STEIN LOOKING AT EXOME MUTATION AND NUMBERS IN VARIOUS CANCER TYPES, THE AAASSOCIATED UPPER TRACK CANCERS FALL RIGHT IN WITH OTHER MUTE O GEN ASSOCIATED CANCERS, TOBACCO ASSOCIATED LUNG CANCERS, UV ASSOCIATED MELANOMAS, SO ABOUT 233 MUTATIONS, MEDIA NUMBER PER EXOME. AND CONTRAST NONARISTOLOCHIC ACID CARCINOMAS FALL RIGHT IN THE MIDDLE OF MOST SOLID TUMORS THAT AREN'T ASSOCIATED WITH MIEWLT O GENS. --MUTE O--MUTOGENS, SO THERE'S SINGLE PAIR BASE SUBSTITUTIONS SUBSTITUTIO NS AND ARE THEY? I DON'T HAVE TO THINK HARDS TO GUESS, THEY ARE T-A TRANSVERSIONS HERE. THIS IS A WAY OF DISPLAYING THE MUTATIONAL SIGNATURE, YOU WILL SEE A LOT OF THIS NEXT MONTH WHEN DR. ALEX ANDROGEN OFFICER TALKS ABOUT MUTATIONAL SIGNATURES HE DEVELOPED THIS WAY OF COMMUNICATING WHAT THE SIGNATURE LOOKS LIKE. THESE ARE THE 4 MAJOR SINGLE BASE CHANGES AND WITH EACH SET THERE ARE 16 HISTOGRAMS AND EACH HISTOGRAM REPRESENTS THE NEIGHBORING NUCLEOTIDE, 16 POSSIBLE COMBINATIONS, YOU WOULDN'T BE ABLE TO READ IT ANYWAY IF YOU PUT IT ON THE AXIS HERE BUT THERE ARE 96 HISTOGRAPS HERE, THIS 1--THIS PEEK HERE IS CAG, SO AND A-MUTATED, AND THIS ADEN O SEEN IS LOCATED IN THE DNA AND THEN THERE ARE REPRODUCIBLE--YOU CAN SEEQUENCE TUMOR AFTER TUMOR AFTER TUMOR AND THESE ARE SEQs ARE MINOR IN THE RATIO. SO CAG IS THE MAJOR NUCLEOTIDE THAT'S--MAJOR TRI NUCLEOTDE WITH THE ACID WITH MINOR CONTRIBUTIONS OF OTHER TRI NUCLEOTIDES AND YOU CAN'T SEE IT HERE BUT THEY'RE USUALLY ON THE 5 PRIME SIDE AND PURINE ON THE 3 PROGRAM SIDE. SO TO SUM UP THE MUTATIONAL SIGNATURE, A-T TRANSVERSES PRE DOMINATE, I TOLD YOU 5-10% IS WHAT YOU TYPICALLY SEE FOR A-T TRANSVERSIONS SO WE DEVELOPED A RULE OF THUMB, ANY TUMOR THAT HAS OVER 20% A-T TRANSVERSIONS WE SUSPECT MIGHT BE CAUSED BY SOMETHING LIKE, IF NOT ARISTOLOCHIC, SOMETHING LIKE IT. THERE'S MARKET STRAND BY AS IN THE GENES, THE MUTATED AD9 IS ALMOST ON THE NONTRANSCRIBED STRAND AND THIS PREFERENCE FOR C-T, AND A-G. NOW THIS IS VERY SIGNIFICANT PREFERENCE AND ACTUALLY MAKES THE BIOLOGICAL CONSEQUENCES MUCH WORSE. CAG IS THE CONSENSUS SEQUENCE FOR TRANSCRIPTIONAL SPLICE SITES. SPLICE ACCEPTOR SITES, SO, IF YOU WANT TO MUTATE A TUMOR SUPPRESSOR GENE AND KNOCK IT OUT, A GREAT WAY TO TO SIT A SPLICE SITE AND REMOVE IT AND THIS WILL EXPLAIN THE ONCOGENIC EFFECTS WITH THE ACID AS IT IS A SPECIALIST AT MUTATEING SPLICE RECEPTOR SITES. SO WITH THIS MUTATIONAL SIGNATURE WE'VE BEEN TALKING ABOUT UPPER TRACT CANCERS BUT YOU CAN ASK, IS THE ACID IS THERE EVERY'RE EVIDENCE THAT'S INVOLVED IN OTHER CANCERS USING THIS MUTATIONAL SIGNATURE AND THIS IS DATA FROM 4 TYPES OF CANCERS. THE FIRST 2 IS FROM OUR SEQUENCING EXPOSURE TO EFFORTS, THE BOTTOM 2 ARE FROM OTHER 1S SO THE UPPER TRACK, URINARY URO THELLIAL CANCER THIS IS CLEAR CELL RENAL CELL CARCINOMA. WE LOOKED AT RENAL CELL CARCINOMA BECAUSE OBVIOUSLY IT CAUSES KIDNEY DAMAGE, NEFF ROUGH ATOM TOXIC AGENT, ALL THOSE CELLS ARE UNDERGOING MUTE O GENESIS AND IN FACT, IT WAS, YOU KNOW THE SIGNATURE IS ALMOST EXACTLY THE SAME AS WHAT'S SEEN IN THE UPPER TRACK CARCINOMAS. THIS HAS ALWAYS BEEN SEEN FOR CLUSTERS NEAR THE ENDEMIC AREA IN EUROPE AND ROMANIA AND IN BOSNIA FOR RENAL CELL CARCINOMAs AS WELL. THERE'S A REPORT FROM CHINESE COHORT OF INTERHEPATITIS EATIC CARCINOMAS SO THIS IS NOW A LIVER CANCER, THIS IS MOVING OUT OF THE KIDNEY IN THE URINARY TRACT TO A WHOLE ANOTHER ORGAN IN EXPERIMENTAL ANIMALS WE WON'T GO INTO A LOT OF DETAILS BUT YOU DO SEE PHENOTYPES AND ALSO DNA ADDUCTS IN THE LIVER IN MICE AND RATS. AS WELL. SO THIS IS PROBABLY NOT A SURPRISE. FINALLY THESE ARE SPORADIC HEPATITIS EAT O CELLIARY CAR NOAM AS FROM THE TC GA SEQUENCING. FROM AMERICAN MAISHTS. NOW THE TCGA AS A COMPONENT OF ASIANS THAT ARE IN THERE AND THOSE CAN BE IDIDN'TIFY EXPTION YOU CAN FIND THE SIGNATURES AMONGST THIS SUBSET, IT IS AMONGST CAUCASIANS AND AFRICAN AMERICANS THAT APPEAR IN THE TCGA. THIS IS NOT A PARTICULARLY GREAT FIT. THE CORRELATION WITH THE UTUC SIGNATURE AND THERE'S A POSSIBILITY THAT THIS IS FROM A DIFFERENT AGENT BUT THEY HAVE MANY--SINCE WE MADE THIS SLIDE, THERE'S BEEN SEVERAL PUBLICATIONS FROM EAST ASIA WHICH NAILS DOWN THE INVOLVEMENT OF THIS MUTATIONAL SIGNATURE IN HEPATITIS EAT O CELLULAR CARCINOMAS. SO TO SUM UP OUR CURRENT KNOWLEDGE OF CANCERS THAT ARISTOLOCHIC MAY BE INVOLVED IN USING THE MUTATIONAL SIGNATURE, THE UPPER TRACK, BLADDERS CANCERS THIS ISN'T A SURPRISE, IT'S A CONTINUOUS URTHELIUM WITH THE UPPER TRACT AND HAS A LOT OF SAME BIOLOGY. RENAL CELL CARCINOMAS I MENTIONED HEPATITIS EAT O CELLULAR CARCINOMAS AND INTERHEPATITIS EATIC COLANGIOC CARCINOMAS. FOUR OUT OF 5 OF THESE HAVE DRIVER GENES, RENAL CARC NOAMS ARE A SPECIAL CASE AND I MIGHT COME BACK AND MENTION THAT AGAIN AT THE END OF THE TALK. WE COULD MAKE AN ARGUMENT FOR MAYBE A DIFFERENT ROLE OF THE ACID IN THE RENAL CELL CARCINOMAS. NOW GO BACK FOR A SECOND. I MENTIONED THIS HEPATITIS EAT O CELLULAR CARCINOMA SIGNAL. THAT LOOKS LIKE THE ARISTOLOCHIC SIGNATURE BUT YOU WOULD LIKE TO HAVE CORROBORATING EVIDENCE OF THIS SO THE OTHER BIOMARKER I'LL BE TALKING ABOUT IS DNA ADDUCTS AND HOW CAN IT BE PUT TOGETHER WITH MUTATIONAL SIGNATURES. VICTORIA [INDISCERNIBLE] LAST MONTH TALKED TO THIS GROUP AND YOU KNOW SHE'S MORE FOCUSED ON THE METABOLIC SIDES OF ARISTOLOCHIC ACID AND SHE PRESENTED THIS SO I WON'T GO OVER IT IN A LOT OF DETAIL BUT IT BASICALLY USHED GOES NITRO REDUCTION IN MANY MAMMALIAN CELLS CAN DO THIS, MANY ORGANISM CANS DO THIS, IT'S DONE PRIMARILY IN THE LIVER AND KIDNEY. HOWEVER, EVEN 1 BLAST AND TISSUE CULTURE CELLS CAN DO THIS. MANY DIFFERENT ENZYMES HAVE SHOWN TO BE ABLE TO UNDERTAKE THIS NATURAL REDUCTION, THOUGH NQO1 IS THE MAIN SUSPECT IN MANY PEOPLE'S MIND HOWEVER, I WOULD LIKE TO POINT OUT THERE ARE HUMAN POLYMORPHISMS THAT HAVE LOW ACTIVITY FOR NQO1 AND WE HAVE GENO TYPED COHORTS OF PEOPLE FROM THE BALKANs FROM THE VILLERAGES AND COHORTS FOR NQL1 AND WE DON'T SEE A CORRELATION SO EVEN THOUGH NQL1 CAN CARRY OUT THIS NITRO REDUCTION, IT'S NOT ESSENTIAL, IT'S NOT REQUIRED, THERE'S A LOT OF CAPACITY IN THE CELLS TO DO THIS NITRO REDUCTION EVENT. VICTORIA SHOWED YOU THAT KAY HAVE THESE INTERMEDIATES WHICH WHEN THEY UNDERGO HYDROLYSIS CAN FORM AN INTERMEDIA THAT CAN REACT WITH A LOT OF DIFFERENT BIOMOLECULES IN THE SALE, DNA INCLUDED BUT THE EXTRA RACYCLICCA MEANS PURINES CAN BE ACTIVE WITH PROTEINS, ET CETERA. OKAY. THERE'S ADDUCTS OF BOTH AD9 AND QUANINE ARE DETECTABLE IN CELLS AND MICE AND EVEN PEOPLE AND RIGHT AFTER EXPOSURE TO THE ACID. SO THE TECHNIQUES FOR LOOKING AT THESE ARE A COUPLE THAT ARE WIDELY USED. ONE POST LABELING WHICH I MENTIONED EARLIER. THIS IS WHERE DNA IS DIGESTED DOWN THE NUCLEOTIDES AND WITH KINASE A P32 IS ADDED, THESE CAN BE SEPARATED BY 2 DIMENSIONAL CHROMATOGRAPHY OR IN OUR CASE A TECHNIQUE PIONEERED BY [INDISCERNIBLE] WHICH USES ELECTROPHORESIS AND WE'RE LUCKY ENOUGH TO HAVE SIPGHTICIZED STANDARDS FROM FRANK JOHNSON'S GROUP HERE AT STONY BROOK AND CHEMISTRY DEPARTMENT AND [INDISCERNIBLE] WHO WORKS WITH HIM. AND YOU CAN FIND, THIS IS 1 OF THE SAMPLES FROM 1 OF THESE SPORADIC PATIENTS HERE IN THE UNITED STATES THAT I HAVE SPOKEN ABOUT. SAMPLES FROM THE KIDNEY CORTEX AND RENAL PELVIS AND WE CAN SEE THAT THESE SAMPLES HAVE AN ADDICT THAT CO-MIGRATES WITH THE ADEN O SEEN ADDUCT OF THE DEOXYADEN O SIGN OF THE DNA. NOW THIS CO-MIGRATES. YOU CAN SEE THERE'S OTHER ADDUCTS IN HERE AS WELL. MOST HUMAN SAMPLES ARE ANIMALS OUT IN THE WILD, WHEN WE CAN GET THESE CLINICAL SAMPLES, WE SEE MULTIPLE ADDUCTS WE SEE A LOT OF THINGS THEY'VE BEEN EXPOSED TO WE WOULD LIKE TO KNOW IF THIS IS THE ARISTOLOCHIC ACID SO ANOTHER TECHNIQUE THAT'S MUCH MORE SPECIFIC IS OUR COLLABORATOR AT THE UNIVERSITY OFIN MIN ROB TERETSKI DOES UPLC, MASS SPEC THE THIRD AND CAN NAIL DOWN THE STRUCTURE OF THESE ADDUCTS. THIS SHOWS THE MS 2 PRODUCT, AGLUE MARIOUS CONE WHICH CAN BE FRAGMENTED AND YOU CAN GET A CHARACTERISTIC SPECTRUM. HE HAS LABELED STANDARDS WHERE WE CAN SPIKE IN THIS AND DO UN EQUIVOCALLY IDENTIFY THESE AND DO THE QUANTITATION. THIS IS INCREDIBLY SENSITIVE METHOD, WHEN THE QUANTITATION IS 5 ADDUCTS PRETENDED WITH THE NUCLEOTIDES THAT CALCULATES OUT TO ON AVERAGE ABOUT 30 ADDUCTS PER CELL IN A DNA DERIVED FROM AND IN A CLINICAL SAMPLE. THIS IS REALLY ROBUST METHOD, CAN YOU TAKE FORMAL PARAFFIN 'EM BEDDED SAMPLES, EXTRACT THE SAMPLE, DNA AND DIGEST IT DOWN AND YOU GET COMPARABLE YIELDS TO FRESH FROZEN SAMPLES. SO THIS IS A REALLY NICE METHOD FOR DOING THE 'EM DEEMIOLOGY. --EPIDEMIOLOGY. SO THIS SLIDE SHOWS A BUNCH OF RESULTS WE ACCUMULATED OVER THE YEARS WITH ROB AND FROM VARIOUS CLINICAL COHORTS COHORTS AND TAIWAN. THREE DIFFERENT CANCER TYPES, YOU KNOW LOOKING IN THE NORMAL TISSUES FROM THESE PATIENTS AND WE CAN FIND THEM READILY DETECTABLE ADDUCTS IN ALMOST ALL OF PATIENTS. ALL OF THE IHEC PATIENTS, INTERHEPATITIS EATIC CARCINOMAS AND ALL THE UPPER TRACK CANCER PATIENTS AND RENAL CELL CARCINOMA PATIENTS. SO THIS IS CONSISTENT WITH THE ALMOST UBIQUITOUS EXPOSURE OF PEOPLE IN TAIWAN TOARISTOLOCHIC ACID. NOW IN THIS CASE WE KNOW THEY WERE'RE WERE EXPOSED WE DON'T KNOW HOW LONG AGO THEY WERE EXPOSED. THIS IS NOT--PEOPLE DON'T KEEP CAREFUL RECORDS OF THEIR HERBAL REMEDIES THAT THEY TAKE AT HOME. WE CAN DO A STUDY THOUGH HOWEVER IN MICE--THIS IS LOOKING AT KIDNEY DNA WHAT HAPPENED WAS WE FED MICE FOR A MONTH WITH A DIET THAT CONTAINED ARISTOLOCHIC ACID AND LET THEM SIT ON THE SHELF AND TOOK MICE AT VARIOUS TIME POINTS AND ASKED WHAT THE ADDUCT LEVELS ARE AND YOU CAN SEE THE LOG SCALE. A NICE LOG LINEAR DECAY IN ADDUCTS BOTH EG ADDUCTS AND THE A-ADDUCTS AND THEY DO DDK. THEY ACCUMULATE AT A LOWER RATE AND THEN DECAY AT A SLIGHTLY FASTER RATE. BUT I HAVE THIS VERY EXPECTATIONS PANNED SCALE THOUGH TO SHOW YOU IF WE LOOK AT HOW SENSITIVE WE CAN ACTUALLY DETECT THESE, OUR LIMIT OF QUANTITATION IS WAY DOWN HERE. YOU CAN EXTRAPOLATE THIS LINE OUT BUT WHERE ARE YOU GOING TO GET DOWN TO LIMIT OF QUANTITATION AND THIS IS 1 YEAR. A MOUSE ONLY LIVES FOR 2 YEARS. SEVERAL MOUSE GENERATIONS BEFORE YOU WOULD EVER GET DOWN TO REMOVING ALL OF THE ADDUCTS OR ADMIN RECORDS ANDUCTS--ADDUCTS WOULD ALL DISAPPEAR. I USED THE WORD REMOVAL, CAN YOU HAVE CELL DIVISION AND DILUTION, CELL DEATH AND ELIMINATION OF CELLS OR YOU CAN HAVE REPAIR OF ADDUCTS, ANDRY MOVAL OF IT FROM THE DNA. THIS IS DATA FROM KIDNEY, SIMILAR DATA IN THE URINARY TRACT, COMPARE THE KIDNEY AND BLADDER ADDUCTS IN THE SAME COHORT OF MICE. WE KNOW 2 TARGET TISSUES BOTH IN MICE AND IN HUMANS. SO WHERE ARE THESE ADUBLGHTS LOCATED? WE HAVE DEVELOPED ANTIBODIES TO THESE ADDUCTS. THESE ARE RAPID MONOCLONAL ANTIBODIES WE USE THE MUTE O GENERATED OF INVITRO REDUCED ARISTOLOCHIC ACID, ADDUCTED DNA AND ALBUMIN AND IT CONDUCTS PROTEIN MOLECULES SO WE DEVELOPED ANTIBODIES AGAINST BOTH AND THE DNA ADDUCTS, AND WE CAN LOOK ON THESE TISSUES, UNTREATED MICE, THESE ANTIBODIES SEE NOTHING. AFTER A MONTH OF EATING THE ARISTOLOCHIC IN THE DIET, YOU CAN SEE ALL OF THE--MANY OF THE NUCLEI IN THE TUBULES IN THE PROXIMAL TUBULES, IN THE KIDNEY SECTION, YOU HAVE COLLECTING DUCTS, YOU HAVE MANY TYPES OF DIEWBULES. YOU CAN SEE THAT THE NUCLEI AND THE PROXIMAL TUBULES ARE HEAVILY STAINED. IF WE WAIT A YEAR, CAN YOU SEE THAT IN--WE CAN DETECT EASILY DETECT ADDUCTS AND OTHERWISE HEALTHY LOOKING CELLS. SO WE BELIEVE THESE ARE STILL LIVING IN CELLS THAT ARE VIABLE IN THE KIDNEY AND NOT REMOVED BY CELL DIVISION OR--WHICH IS NOT VERY--THE RENAL CORTEX JUST DOESN'T REALLY PROLIFERATE ALL THAT MUCH. SO PERHAPS AFTER WOUNDING THERE MIGHT BE REPLACEMENT BUT THERE ISN'T A LOT OF CELL DIVISION GOING ON AND WE DON'T THINK THERE'S A LOT OF CELL DEATH GOING ON IN THIS CASE. THE BEST STUDY IN HUMAN SAMPLES WAS DONE BY [INDISCERNIBLE] ONCE AGAIN WORKING WITH THE BELGIAN COHORT SO 11 BELGIAN PATIENTS WHICH EVENTUALLY HAD THEIR KIDNEYS REMOVED OVER A LONG TIME, NOT INITIALLY IN THE INITIAL BURSTS OF THE TIME LAPSE, TIME HAD PASSED. TIME OF EXPOSURE TO EXPOSE TOWER SURGERY, RANGED FROM, YOU 99 IN COHORT 10-20 YEARS SO 1-2 DECADES. HE USED POST LABELING ONCE AGAIN BUT FOUND 11 OUT OF 11 PATIENTS HAD ADDUCTS. THESE ARE DA-AL1 ADDUCTS AND INITIAL LOAMACYY WE CAN FIND FOR EVIDENCE FOR 4 TYPES OF ADDUCTS BUT THERE ARE OTHER TYPES OF ARISTOLOCHIC ACIDS IN AN ARISTOLOCHIC SAMPLE AND THERE'S AAC1 AND AA2 AND THE ADDUCTS DGAL1, DGAL2, AAL1, AND DALL2, AND HOWEVER AFTER 1020 YEARS ONLY THE DA-AL1 ADDUCTS PERSISTED SO THESE SOMEHOW FIND AIAN WAY OF--FIND A WAY OF STAYING IN THE DNA. SO TO TRY TO EXPLAIN THIS, VICTORIA A WHILE AGO, WORKING ON THIS EVIDENCE THAT WE KNEW THAT TRANSCRIBE STAND WAS YOU KNOW ADDUCTS WERE--MUTATIONS DID NOT APPEAR ON THE TRANSCRIBED STRAND; YOU KNOW ASSUME THAT TRANSCRIPTION COUPLE REPAIR WHERE POLYMER ACE COMES ALONG, HITS AN ADDUCT AND STOPS AND WILL HIT THE SECTION OF THE DNA AS WE MOVE THROUGH NUCLEOTIDE EXCISION REPAIR, IS PROBABLY THE EXPLANATION FOR THIS HOWEVER ON THE OTHER STRAND, WHERE NORMALLY GLOBAL GENOME REPAIR WOULD REMOVE SUCH A BULKY ADDUCT BECAUSE THE DNA STRUCTURE IS PERTURBED IN SOME WAY THIS DIDN'T HAPPEN AND WHAT VICTORIA SHOWED WAS THAT SEVERAL COMPONENTS UNIQUE TO TRANSCRIPTION COUPLE REPAIR, IF YOU HAVE CELL LINES, MUTATED AND THEN YOU CREATE VERY SENSITIVE--CELLS ARE SENSITIVE TO ARISTOLOCHIC ACID HOWEVER XPC CELL LINES WHICH ARE DEFECTING GLOBAL GENOME REPAIR, YOU KNOW YOU STILL SHOULD--YOU DIDN'T EFFECT THE SENSITIVITY. FRACTORS THAT ARE COMMON TO BOTH HE'S PATHWAYS WOULD INCREASE THE SENSITIVITY AS WELL. SO CONCLUSION IS THAT THESE ADDUCTS WHICH AREN'T REPAIRED BY GLOBAL DENOAM REPAIR, THEY ARE INVISIBE, THEY SHOULD BE REPAIRED BUT THEY'RE NOT. CARLOS DELOS SANTOS HERE AT STONY BROOK DID STRUCTURES WITH SYNTHESIZING HERE AT STONY BROOK AND HE FOUND IN SOME SEQUENCE CONTEXT ACCIDENT VERY LITTLE PROTERBATION OF THESE STRUCTURES ON THESE ADDUCTS. SO THESE OBSERVATION COULD EXPLAIN WHY THESE ESCAPE GLOBAL GENOME REPAIR. SO PUTTING ALL THESE TOGETHER, WE CAN MAKE A MODEL FOR THIS REPAIR. OF COURSE, YOU KNOW YOU GET ARISTOLOCHIC, ADDUCT, IN THE DNA, HERE WITH AT BASE PAIR, TRANSCRIPTION COUPLE REPAIR, THIS HAPPENS ON A TRANSCRIBED GENE AND STRAND YOU WOULD GET IMMEDIATE REMOVAL AND REPAIR. AND BACK TO WILD TYPE SITUATION, HOWEVER IF THIS IS ON--BETWEEN GENES SOME PLACE OR ON THE NONTRANSCRIBED STRAND, AT LEAST A SUBSET OF THESE ADDUCTS WILL ESCAPE GLOBAL GENOME REPAIR AND PERSIST IN THE DNA. NOW IF SOMETHING HAPPENS WHERE THE CELL HAS TO REPLICATE FOR NORMAL HOMEOSTASIS, THIS IS IN A STEM CELL, OR IF THERE'S WOUNDING AND YOU HAVE TO REPLICATE CELLS IN ORDER TO REPAIR A TISSUE AND UNDERGO S-PHASE, YOU HAVE CHOICES, YOU HAVE FAITHFUL TRANSLATION SYNTHESIS, OR UNFAITHFUL. NOW [INDISCERNIBLE] HERE AT STONY BROOK STUDIED THIS AND FOUND THAT THIS IS LIKE A 50/50 CHOITION. YOU CAN SPLIP A COIN. ABOUT HALF THE TIME YOU WILL HAVE TRANSLAIKS SERVICES INSERTION OF A T ON THE RESIDUE AND YOU WILL GO BACK TO STATUS QUO. YOU WILL HAVE A CELL WHICH WILL NOW HAVE AN ADDUCT THAT WILL PERSIST AND GO BACK TO THE PREEXISTING SITUATION. IF YOU HAVE UNFAITHFUL TRANSLATION SYNTHESIS HOWEVER, YOU NOW HAVE A MISMATCH AND NOW YOU DO HAVE A SUBJECT, SUBSTRATE FOR GLOBAL GENOME REPAIR. VICTORIA SHOWED THAT MISMATCHES AND BUBBLED ARE SUBJECT FOR GLOBAL GENOME REPAIR. NOW MANY PEOPLE HAVE SHOWN THERE'S ARREST ASSOCIATED WITH TREATING CELL CULTURES WITH THE ARISTOLOCHIC ACID SO THERE'S THE MODEL WHERE YOU HAVE ONGOING REPAIR AFTER DNA REPLICATION. OKAY. NOW, THIS ARE IS REPAIR BUT IT'S NOT REALLY REPAIR, THIS IS MISS IS A MISMATCH AND YOU ARE REMOVING THIS ADDUCT AND INSERTING A T OPPOSITE--YOU'VE GOT FROM A-T, TO T-A SO IN 1 CELL CYCLE YOU CREATE THE MUTATION AND YOU FIX IT IN THE DNA AND SO THIS IS ACTUALLY BAD. THIS IS OKAY, BUT IT MAY BE BAD AS WELL. BECAUSE THE STATUS QUO IS MAINTAINED IF YOU HAVE TO DIVIDE AGAIN, YOU CAN THEN HAVE THIS 50/50 CHANCE THAT YOU ARE NOW GOING TO GENERATE A MUTATION. IF THIS ADDUCT IS LIVING IN A TUMOR SUPPRESSOR GENE SOONER OR LATER YOU'RE GOING TO CAUSE A MUTATION. IF THIS IS AT A CRUCIAL SPOT IN AN ONCA GENE, SOONER OR LATER IT WILL CAUSE A MUTATION SO PEOPLE WHO HAVE ADDUCTS IN THEIR DNA IF THEY ESCAPE THE FIRST WAVE OF THE RENAL, YOU KNOW RENAL DISEASE, THEY WILL BE LIVING WITH THESE ADDUCTS FOR THE REST OF THEIR LIFE AND EVENTUALLY THESE MAY ACTUALLY BE INITIATE ING CANCER. SO TO SUMMARIZE THESE ADDUCTS, WE ARE VERY HAPPY TO HAVE THEM BECAUSE THEY'RE EXCELLENT BY O MARKERS. WE CAN DETECT THEM AT VERY LOW LEVELS. EXTREMELY LOW LEVELS AND THEY PERSIST FOR A LONG, LONG TIME. YOU KNOW THIS--FOR US AS RESEARCHERS, WE'RE DELIGHTED. HOWEVER, THESE SAME AS I JUST MENTIONED THESE SAME PROPERTIES MAY MAKE THEM AN ONGOING HEALTH HAZARD FOR PEOPLE WHO ARE HARBORING ADDUCTS IN THEIR DNA SO VERY EXPOSE TOWER ARISTOLOCHIC ACID EARLY IN LIFE MAY HAVE CONSEQUENCES MUCH, MUCH, LATER. SO TO PUT THESE 2 LINES OF EVIDENCE TOGETHER AND THESE 2 CORROBORATING BIOMARKERS TOGETHER IN THESE VARIOUS CANCERS, WE HAVE A LOT OF EVIDENCE FROM THE STANDARD EPIDEMIOLOGY THAT ARISTOLOCHIC ACID IS ASSOCIATED WITH UREATAL CANCERS AND THIS IS CORROBORATED BY THE MOLECULAR EPIDEMIOLOGY, WE FIND THESE SIGNATURE THAT ARE EASY TO PROTECT AND THEY PREDOMAIN NATIVE AMERICANS IN THE NORTHERN THE GENOMIC LANDSCAPE OF THESE CANCERS FROM THESE PATIENTS. AND PROBABLY VERY IMPORTANTLY THE MUTATIONS ARE IN DRIVER GENES. I SHOWED YOU THE TP53 GENE, OTHER DRIVER GENES FOR BLADDER CANCERS AND THERE A-T MUTE O GENESIS AS WELL AND THIS HAS BEEN MANY PUBLICATIONS FROM OUR GROUP AND MANY OTHERS. I MENTIONED RENAL CELL CARCINOMA. THERE WAS A SPECTACULAR PAPER LOOKING AT THE GENOMICS OF RENAL CELL CARCINOMA IN EUROPE A FEW YEARS AGO, THEY HAD COHORTS FROM ENGLAND, POLAND FROM RUSSIA AND FROM ROMANIA. AND ROMANIA, ALL OF THE TUMORS ARE AT LEAST OF THE 12 OR 14 TUMORS JUST OUT OF THE BLUE HAD MUTATIONAL SIGNATURES OF ARISTOLOCHIC ACID. THE OTHER COHORTS FROM OTHER PARTS OF EUROPE WERE TYPICAL OF RENAL CELL CARCINOMAS SEQUENCED AROUND THE WORLD. SO, THIS--WE SEQUENCED RENAL CELL CARCINOMAS IN TAIWAN WITH THE SAME RESULTS SO MUTATIONAL SIGNATURES PRESENT IN RENAL CELL CARCINOMAS FROM 2 EXPOSED POPULATIONS. ADDUCTS ARE THERE, WE'VE DETECTED ADDUCTS IN THESE PEASHTS AND ALMOST ALL OF THE PATIENTS AS WELL. WE'VE ALSO DONE EPIDEMIOLOGY, AND I MENTIONED EARLY EARLY IN THE TALK, THE DEPRESCRIPTION GATTA BASE IN TAIWAN, THIS IS A REALLY USEFUL RESEARCH TOOL, TAIWAN RESEARCHERS CAN APPLY FOR PERMISSION TO EXAMINE THE DATABASE IN LIMITED WAYS AND THEY CAN LOOK AT PRESCRIPTIONS PEOPLE HAVE HAD, AND YOU CAN SHOW THERE'S A POSITIVE CORRELATION WITH DOSAGE OF ARISTOLOCHIC ACID THAT PEOPLE PRESCRIBED AND THE CURRENCE OF RENAL CELL CARCINOMA. HOWEVER FWE SEQUENCE MANY OF THESE GENOMES NOW, AND WE REALLY DON'T SEE A-T MUTE O GENESIS IN DRIVER GENES. YOU KNOW WE LOOKED AT THE VHL GENES AND DR. WANG OVER HERE IN ANOTHER LAB NOW BUT WHEN SHE WAS ROTATED IN MY LAB SEQUENCED VHB GENES FROM THE CARCINOMAS AND WE'VE DONE THE SEQUENCING AND OTHER PEOPLE HAVE DONE THE SEQUENCING IT'S SUSCEPTIBLE TO A-T MUTE O GENESIS AND LARGE PROPORTIONS AX ROUND THE WORLD ARE ATELEVISION H, HOWEVER THERE'S NO ENRICHMENT OF THE ARISTOLOCHIC ASSOCIATED FOR A-T MUTE O GENESIS IN THE VHL GENE. SO THERE'S ARISTOLOCHIC IS PLAYING A DIFFERENT ROLE IN THE ETIOLOGY OF THESE. IT'S CONSISTENTLY FOUND TO HAVE BEEN PRESENT. PEOPLE HAVE BEEN EXPOSED TO IT AND WHAT WE THINK IS GOING ON, IS STOL SOL IS CAUSING SUCH GREAT TISSUE DAMAGE IN THE KIDNEYS OF THESE PATIENTS THAT THERE'S A LOT OF REPLICATION CELLS AND NORMALLY DOESN'T HAPPEN, AND SO NASCENT TUMORS WHICH MIGHT NEVER FORM WILL GO ON TO FORM. THEY HAVE AN OPPORTUNITY TO PROLIFERATE AND WE THINK THAT'S WHAT HAPPENED IN THAT CASE. HEPATITIS EAT O CELLULAR CARCINOMA I SHOULD MENTION SPECIFICALLY, THIS IS NOT OUR WORK, SPECIFICALLY FOR MUTATIONAL SIGNATURE, THIS WAS DONE SEVERAL OTHER GROUPS IN JAPAN AND CHINESE COHORTS. BUT WE HAVE LOOKED AT ADDUCTS AND JAPANESE COHORT AND THERE ARE 10% OF THE PEOPLE WHO ARE EXPOSED TO ARISTOLOCHIC ACID IN THAT POPULATION. HEPATITISHEPATITIS HEPATITIS EAT O CELLULA R CARCINOMA WAS NUMBER 1 IN THE WORLD, NOT SO MUCH IN THE UNITED STATES BUT THROUGHOUT ASIA. THERE ARE MANY CAUSES OF HEPATITIS E HEPATOCYTE AT O CELLULAR CARCINOMA FROM THE SIGNATURE PRESENT WE COULD ARGUE THAT HEPATITIS EAT O CELLULAR CARCINOMAS ARE ASSOCIATE WIDE THE ARISTOLOCHIC ACID SO YOU COULD ELIMINATE OVERNIGHT 10-15% OF A COMMON CANCER BY STOPPING THE ARISTOLOCHIC ACID THAT'S A SIGNIFICANT CONTRIBUTION TO PUBLIC HEALTH, I THINK. THIS LAST CANCER INTERHEPATOCYTEATIC CO LANCHIA CARCINOMA IS CANCER OF THE BIODUCTS WITHIN THE LIVER. IT'S ALMOST ALWAYS FATAL. SO IT'S A VERY SERIOUS CANCER, STUDIES FROM CHINA IN THE COHORT FROM THE SHANGHAI AREA, TOWED SHANKAR ONCE AGAIN 10-15% OF THOSE PATIENTS HAD THE MUTATIONAL SIGNATURE, HAD SIGNATURE IN DRIVER GENES. WE ARE IN THE MIDDLE OF A STUDY OF TAIWANESE IHEC PATIENTS AND AT LEAST SO FAR WE ARE NOT SEEING THE MUTATIONAL SIGNATURE. SO THERE'S A DIFFERENT THERE. IT COULD BE THE SIZE OF OUR COHORT IS TOO SMALL, WE'RE LOOKING AT 10-15 WE MIGHT HAVE TO EXPAND OUR COHORT BUT IN OUR& MIND THE JURY IS STILL OUT ON THIS CANCER. HOWEVER IN THE REPORT, IN THE LITERATURE, THERE IS DEFINITELY DRIVER MUTATIONS AND A-T MUTATIONS AND MUTATIONAL SIGNATURES PRESENT THROUGHOUT THE GENOME, AND I SHOWED YOU DATA EARLIER WHERE WE'VE LOOKED, IHEC PATIENTS HAVE THE ADDUCTS AS L. SO TO SUMMARIZE THEN, THERE'S AN EVER- EXPANDING UNIVERSE OF HUMAN DISEASES WHICH ARE ASSOCIATED WITH THE ARISTOLOCHIC ACID. JUST 20 YEARS AGO WE HAD NO CLUE THAT ARISTOLOCHIC WAS A PUBLIC HEALTH CONCERN, NOW WE APPRECIATE THAT IT'S A MAJOR CAUSE OF UROTHELIAL, LIVER AND PERHAPS [INDISCERNIBLE] TUMORS AS WELL. THESE ARE MARKETED BY HIGH MUTATION RATE, A UNIQUE SIGNATURE THAT'S TURNED OUT TO BE A NICE TOOL FOR DETECTING THESE. AND I TALK ABOUT THE ADDUCTS AND I THINK THE ADDUCTS ARE PARTICULARLY INTERESTING, THEY'RE A GREAT TOOL, IN THE DNA REPAIR CONTEXT, MAKE GLOBAL GENOME REPAIR AND THAT MIGHT BE ACTUALLY UNDERLYING OF WHY THIS IS SUCH A DANGEROUS CAR SIN CARCINOGEN. SOME CURRENT QUESTIONS WE'RE ADDRESSING ARE WHETHER THESE PERSISTENT DNA ADDUCTS ARE AN ONGOING SOURCE OF MUTATIONS. WE'RE USING OUR ANTIBODIES TO PURIFY DNA FROM--THAT HAS BEEN TREATED WITH ARISTOLOCHIC ACID, ALLOWED TIME FOR REPAIR AND REMOVAL OF ADDUCTS AND WE'RE-WE WANT TO SEQUENCE THOSE AND MAKE LIBRARIES AND SEQUENCE THOSE AND SEE WHERE THESE ADDUCTS ARE. ARE THEY DISTRIBUTED IN THE GENOME? IN THE TUMOR SUPPRESSOR GENES WAITING LIKE BOMBS TO GO OFF? OR ARE THEY IN PARTS OF GENOME THAT MIGHT NOT BE SURVEILLED SO MUCH? PLACES WHERE WE DON'T WORRY, CAN WE PUT PEOPLE'S MIND AT EASE OR WE HAVE TO ACTUALLY ADDRESS THIS AS A SURVEILL OF PEOPLE WHO HAVE ADDUCTS MORE OFTEN FOR DEVELOPMENT OF CANCERS TO TRY TO CATCH THEM EARLY. OKAY, SO THE CONTRIBUTORS ARE, YOU KNOW A LOT OF THEM HERE ON THE SLIDE IS MANY MORE, ARTHUR GROMAN HAS PUT TOGETHER A GREAT GROUP HERE AT STONY BROOK WHICH FOCUSED ON THIS PROSPECT. I WANT TO MENTION PARTICULARLY, YOU HEARD HER NAME VICTORIA [INDISCERNIBLE], YOU HEARD HER TALK LAST WEEK, SHE'S HAS BEEN INVOLVED IN ALMOST ALL THE ASPECTS IN THE METABOLIC SIDE AND THE DNA REPAIR SIDE OF THESE STUDIES AND KATIE [INDISCERNIBLE] IS ASSISTING IN THIS EFFORT TO TRY TO FIND WHERE THESE ADDUCTS ARE EXISTING IN THE GENOME. THAT HAVE ESCAPED REPAIR. WE'VE DONE A LOT OF WORK WITH BERT VOGELSTEIN'S GROUP AT JOHNS HOPKINS, ALL THE SEQUENCING WE HAVE DONE WITH THEM AND WE'RE FORTUNATE THAT THEY'RE INTERESTED IN THIS PROBLEMS AND WE DISCUSS THESE ALL THE TIME WITH THEM AS WELL. GOT GREAT CLINICAL COLLABORATORS IN TAIWAN AT NATIONAL TAIWAN UNIVERSITY. THEY HAVE THE MISFORTUNE OF HAVING A LOT OF PATIENTS COME IN PRESENTING WITH THESE UPPER TRACK CANCERS, BLADDER CANCERS, RENAL CELL CARCINOMAS. WE HAVE THE GOOD FORTUNE WE'RE COLLABORATING WITH THEM AND WE HAVE A NEVER ENDING STREAM OF CLINICAL SAMPLES. WE HAVE GREAT COLLABORATIONS IN CROATIA, WITH BOJAN JEWELL, AKOVIC, WITH THE BALCANS AND THOSE AT UNIVERSITY OF MINNESOTA WITH MASS SPEC METHODS AND ROB AND I HAVE A COPI-GRANT TO ADD GENOMICS TO EXTEND THIS TO MORE THAN 1 ADDUCT AS A TIME IF WE CAN SAME ULTIMATELY TAINIOUSLY ASSESS EXPOSURES TO MANY THINGS IN PATIENTS AT THE SAME TIME. OKAY, SO I'LL STOP THERE AND I GUESS--OKAYS. >> ANY QUESTIONS? >> YES, THANK YOU. THAT'S VERY NICE. WE WILL GO AROUND TO THE DIFFERENT SITES, EACH SITE CAN ASK 1 QUESTION. AND THEN WE'LL GO TO THE NEXT 1. LET'S GO--LET'S GO TO BALTIMORE. PLEASE MUTE AT ALL THE OTHER SITES. >> THAT WAS A VERY NICE PRESENTATION. I HAVE A QUESTION FOR YOU. HOW DO YOU SUPPOSE THAT THIS WAS USED FOR 2000 YEARS OR MORE WITHOUT SOMEBODY REALIZING IT'S A PROBLEM UNTIL JUST RECEIPTLY? >> WELL, CANCER TAKES YOU KNOW DECADES SOMETIMES TO DEVELOP AND IF THE IMMEDIATE MALADY IS TAKEN CARE OF AND THEY LEAVE, YOU DON'T SEE THEM--YOU KNOW SOMETHING HAPPENS YOU KNOW 20 YEARS LATER, YOU KNOW MAKING THE CONNECTIONS BECAUSE IT'S QUITE DIFFICULT. AS FAR AS THE RENAL FAILURE, I WOULD IMAGINE THAT PROBABLY WAS KNOWN TO SOME EXTENT. WOE HAVE ANN ECTOMYOSIN DETOMORROW NATION FROM THE VETERINARY WORLD IN CROATIA WHERE THE VETS MADE THAT CONNECTION AND THEY KNEW HORSES THAT WERE FED, HAY THAT WAS CONTOMINATED WITH THE ARISTOLOCHIC WOULD LEAD TO RENAL PROBLEMS. THEY APPRECIATED THAT AND THEY JUST TOLD THEIR FARMERS, YOU KNOW, AND THE CALVARY AND THE ARMY WE DON'T--DO NOT--MAKE SURE THE HAY IS CLEANED UP A LITTLE BIT. SO, YOU KNOW ALL OF THOSE TYPE OF FACTORS CONTRIBUTE. >> OKAY, AND THANK YOU. PLEASE BE SURE TO MUTE AT ALL THE OTHER SITES. LET'S GO TO NIEHS. >> DR. ROSENQUIST, THANK YOU SO MUCH FOR A NICE TALK. REALLY LOVELY MADE WE WONDER THOUGH, DO YOU GET AA-ADDUCTS IN THE PLANT DNA OR DO NAY NOT METABOLICALLY ACTIVATE THIS STUFF? >> I HAVE NOT LOOKED AT THE PLANT DNA, WE CAN CERTAINLY DO THAT. SEVERAL--NOW OBVIOUSLY ARISTOLOCHIC PLAYS A ROLE IN THE BIOLOGY OR IT WOULDN'T HAVE BEEN SELECTED. THERE ARE ACTUALLY SPECIES OF BUTTERFLIES THAT USE ARISTOLOCHIC AND CONCENTRATE THE ARISTOLOCHIC ACID AND CAN EVADE THE NEGATIVE CONSEQUENCES SO YOU KNOW, THEY SEQUESTER IT, THEY'RE BRIGHT ORANGE, THE CAT CATERPILLARS ARE BRIGHT ORANGE, DON'T EAT ME THEY'RE FILLED WITH THE ARISTOLOCHIC SO THERE'S A BIOLOGICAL USE FOR THESE. SO IT IS POSSIBLE FOR, YOU KNOW FOR EVOLUTION TO HAVE SELECTED FOR SEQUESTERING THE ARISTOLOCHIC AND KEEPING IT AWAY FROM THE GENOME OF THE HOST THAT IS USING IT. >> YEAH IT'S JUST VERY INTRIGUE, IF THEY HAVE A WAY TO REMOVE IT, IT WOULD BE NICE TO KNOW ABOUT IT. THANK YOU VERY MUCH. >> OKAY, PLEASE MUTE, AT THE RESEARCH TRIANGLE, LET'S GO TO PORTLAND, OREGON. >> TOM JUST ABSOLUTELY FANTASTIC STORY AND CONGRATULATIONS TO THE ENTIRE STUDY GROUP, ARTHUR I KNOW, THIS HAS BEENINARY AND DEAR TO YOUR HEART FOR A WHILE, WE LOVE TEACH THANKSGIVING TO OUR GRADUATE STUDENTS. THE QUESTION THAT I HAD WAS IN CONTRAST TO HAPLOTOXINS AND WHICH YOU REALLY DON'T SEE MUCH INLET WAY OF KIDNEY OR URTHELLIAL TIME CANCERS AND I APOLOGIZE, DIDN'T HEAR VICTORIA'S TALK RELATIVE TO THE METABOLISM BUT IN TERM TURN DO YOU SEE THE MEDICAL ACTIVATION OCCURRING IN THE KIDNEY AND THAT THIS IS WHAT IT'S GIVING THE KIDNEY IN THE UPPER UROTHELIAL TRACT CANCERS OR IN AND ALSO WHEN YOU'RE DOING YOUR IMMUNE O HISTOCHEMISTRY STAINING, DID YOU TAKE A ELECTRIC AT THE LIVER TO SEE WHETHER OR NOT THE CLEARANCE OR FORMATION AND CLEARANCE FROM THE LIVER HAD A DIFFERENT KINETIC VERSUS HAVING THE KIDNEY? >> YEAH, CAN YOU SEE THE ADUCS IN BOTH THE LIVER AND IN THE KIDNEY. WE DID NOT DO THIS WORK BUT RAN IN BEIJING DID THE PHARMA CO KINETICS AND I DID CAREFUL STUDIES AND YOU CAN SEE ARISTOLOCHIC ACID ACCUMULATING IN THE KIDNEY SO IT'S NOT ALL METABOLIZED IN THE LIVER AND WE KNOW THAT METABOLISM CAN HAPPEN IN THE KIDNEY. SO MICE THAT YOU CAN TAKE KIDNEY SLICES, CAN YOU DIRECTLY TREAT THEM OR DO PROFUSE KIDNEYS AND SO, KID FLEES ARE PERFECTLY COMPETENT IN TAKING UP THE ARISTOLOCHIC ACID AND METABOLIZING IT AND CAUSING THE--AT LEAST THE RENAL DISEASE AND ALSO THE ADDUCTS, I DIDN'T GO INTO THIS, IN THIS TALK, WE HAVE REASON TO BELIEVE THAT RENAL DISEASE AND THE CANCER IS 2 DIFFERENT PROCESSES AND THE ADDUCTION OF DNA IS NOT THE DRIVING FORCE OF THE RENAL DISEASE, WE THINK SOMETHING ELSE IS GOING ON THERE, I DIDN'T GONE INTO THAT AT ALL. BUT AT THE SAME TIME, IT'S ALSO HAPPENING IN THE LIVER. LIVER, ADDUCTS ACCUMULATE IN THE LIVER WHICH AGER THAT THE METABOLISM IS OCCURRING THERE AS WELL, IN THE UROTHELIUM OF THE BLADDER AND YOU CAN ALSO GET TO A MUCH LOWER EXTENT AND LONG TISSUE IN THERE. BUT THE REAL KIDNEY AND THE LIVER AND BLADDER ARE 3 TISSUES THAT HAVE THE MOST ADDUCTS AT LEAST IN A RODENT, IN A MOUSE. >> THANK YOU VERY MUCH. >> ROOK, COULD YOU PLEASE MUTE WHEN SOMEBODY ELSE IS TALKING I THINK THAT MAY BE WHILE WE'RE GETTING FEEDBACK. >> LET'S GO TO LEXINGTON, 1010. --KENTUCKY. IS ANYONE IN KENTUCKY? >> OKAY, IF NOT LET'S GO TO UNIVERSITY OF GAINSVILLE, FLORIDA. >> HI THERE, THAT WAS A GREAT TALK, TOM. STATE OF EMERGENCY WONDERING IS THERE A PROPENSE URETER FOR P53 OR YOU KNOW WHAT ARE YOURDDUCTS- THOUGHTS ON WHY YOU DETECT P53? CAN YOU HEAR ME? >> OKAY, YOU HAVE TO UNMUTE WHEN YOU WANT TO TALK. >> YES WE DID. >> OH AT STONY. >> CAN YOU HEAR ME NOW? >> YES. >> YES. >> OKAY. SO, I DON'T THINK THERE'S ANYTHING SPECIAL ABOUT P53 ITSELF. I THINK THESE MUTATIONS ARE HAPPENING THROUGHOUT THE GENOME. IT JUST--IN URTHELLIAL CANCER P53 IS 1 OF THE MAJOR DRIVER GENES. THAT'S WHY WE RECOVER IT. YOU KNOW IF YOU STUDY URTHELLIAL CANCERS ANYWHERE IN THE WORLD YOU WILL FIND THAT HALF OF THEM HAVE P53 MUTATIONS. I THINK THAT THE FACT THAT P53 IS TUMOR SUPPRESSOR GENE AND HAS MANY SPLICE JUNCTIONS THAT CAN BE HIT BY THIS MUTE O GENERATED, I MEAN A LOT OF OTHER AMINO ACID CHANGES AS WELL, BUT A HIGH PROPORTION ARE AT SPLICE JUNCTIONS SO YOU CAN JUST KNOCK IT OUT UNEQUIV EQUIVOCALLY ON THESE SPLICE JUNCTIONS SO THAT IS 1 OF THE THINGS THAT ARE MAKING IT SUCH A PARTICULAR POTENT CARCINOGEN FOR TP53 DRIVEN CANCERS. >> SO IF YOU--YOU MENTIONED THAT YOU CAN DETECT THESE MUTATIONS OR ADDUCTS IN LUNGS FOR EXAMPLE, as well, so in lungs can you see it in how other places as well. >> AT A MUCH LOWER LEVEL. >> YES, OKAY. THANK YOU. >> OKAY. PLEASE MUTE IN FLORIDA. IS THERE ANYONE THERE IN MOBILE, ALABAMA? >> NO? OKAY, PLEASE MUTE TEMPORARILY. I HAVE A QUESTION HERE. I'M VERY INTRIGUED BY THE PERSISTENCE OF THE ADDUCTS OVER SUCH A LONG TIME. I WONDER IF THEY CAN BE PRESENT IN STEM CELLS OF THE GUT FOR INSTANCE OR OTHER OR THE BONE MARROW? OR EVEN MORE CONCERNING IN THE OVARIES? AND IF THERE'S ANY EFFECTS FROM 1 GENERATION TO THE NEXT IN THE MICE? >> DID I UNMUTE SUCCESSFULLY, CAN YOU HEAR ME? >> YES. >> OKAY. THOSE ARE ALL GREAT QUESTIONS THAT WE HAVEN'T REALLY LOOKED AT IN DETAIL. YOU'RE NOT THE FIRST 1 TO PROPOSE THIS MIGHT BE A MOUSE MUTA GENERATED, HOWEVER WHETHER THE GERM CELLS ARE--WE DON'T REALLY SEE A HIGH LEVEL OF ADDUCTS THERE WE HAVEN'T PURSUED IT WOULD BE A CONCERN. THOUGH IT IS CONCERNING TO THINK ABOUT IT. AND THEN THE OTHER TISSUES WE HAVEN'T DONE THE WORK. WE HAVEN'T DONE THAT. >> OF THE TREATED MICE HAVE YOU CHECKED TO SEE IF THEY'RE FETTERILE, ANY PROBLEMS WITH THE LITTERS THEY GAVE BIRTH TO? YOU DON'T NEED TO LOOK AT THE ADDUCTS YOU COULD JUST SEE THE EFFECTS? >> NO, WE HAVEN'T DONE THAT. WE HAVEN'T TREATED THEM AND LOOK OVER GENERATIONS. YOU KNOW, OR TO PUT THEM IN SOME TYPE OF--THERE ARE PANELS, YOU KNOW MICE THAT SPECIFICALLY HAVE A PORTERS THAT YOU CAN DETECT MUTATIONS. WE HAVEN'T DONE ANY OF THAT. >> WELL THERE'S ANOTHER GROUP. IF THERE ARE ANY QUESTIONS AT ANY OF THE OTHER SITES YOU CAN JUMP IN AGAIN? >> WE HAVE A QUESTION HERE IN BALTIMORE. >> OKAY. >> THANK YOU FOR YOUR TALK, THAT'S 1 OF THE BEST DNA REPAIR SPEECHES I'VE HEARD IN A WHILE MY QUESTION TO YOU WAS HAVE WE COMPARED THE MUTATIONAL PROFILE OF ARISTOLOCHIC TO OTHERS WITH SIMILAR STRUCTURE? >> COULD YOU SPEAK UP? >> YEAH, SO MY QUESTION WAS HAVE WE COMPARED THE MUTATIONAL PROFILE OF ARISTOLOCHIC ACID TO THOSE THAT HAVE SIMILAR STRUCTURES? >> YOU MENTIONED THE MUTATIONAL SIGNATURES NATURE IS QUITE UNIQUE. AND HASN'T BEEN SEEN BEFORE, THAT SPECIFIC PATTERN OF 96, YOU KNOW TRI NUCLEOTIDES IN THE SORT OF--YOU KNOW LAND SCAPE PICTURE THAT I PUT UP, THE WAY OF LOOKING AT IT, I THINK THAT WOULD JUMP OUT AT YOU, THERE ARE A FEW OTHER A-T MUTE O GENERATEDS BUT THEY DON'T HAVE THAT TYPE OF PROFILE. THAT'S NOT TO SAY THAT IT CAN'T BE SOME OTHER MUTE O GEN THAT EFFECTS IT AND AD9S AND BULLETY PLUTO GEN AND STACK IN THE HELIXESOMEULARLY AND GIVE THE SAME TYPE OF MUTATIONAL SIGNATURE BUT IT IS POSSIBLE TO TELL THE OTHER KNOWN A-T MUTE O GENERATEDS THAT THEY ARE DIFFERENT IN THEIR GENOME WIDE MUTATIONAL SIGNATURE. >> YEAH, I THOUGHT IT WAS INTERESTING TO SEE THE--HOW IT TARGETS REGIONS OF USE WHERE YOU SHOW [INDISCERNIBLE]. >> ONCE AGAIN I CAN'T CATCH THAT. >> YES, SOPHISTICATED SO I WAS INTERESTED WHEN YOU SHOWED THAT SLIDE, HOW IT TARGETS THE UNTRANSCRIBED REGIONS SO IF THAT WAS A GENERAL THING FOR OTHER MUTE O GENERATEDS THAT ARE SIMILAR TO ARISTOLOCHIC ACID? >> TARGETING THE NONTRANSCRIBED REGIONS? >> YEAH. YOU JUST PASSED IT. YEAH. THAT 1. SNR YOU REFERRING TO OUT HERE? >> RIGHT. >> WELL I DON'T KNOW IF I WOULD CALL THAT TARGETING. YOU KNOW IT'S--YOU KNOW A SMALL NUMBER OF THE MUTATIONS, THIS IS PROBABLY--I WOULD IMAGINE THIS IS PROBABLY FROM MY--YOU KNOW I SOMETIMES WE GET MULTIPLE MUTATIONS IN A PATIENT AND I WOULD IMAGINE THIS PROBABLY IS SORT OF A PASSENGER MUTATION THAT CAME ALONG FOR THE RIDE. I CAN GO BACK AND CHECK, BUT YOU KNOW I WOULD--THAT WOULD BE MY FIRST GUESS THERE. >> OKAY, THANK YOU. >> OKAY THANK YOU. ARE THERE QUESTIONS AT ANY OF THE OTHER SITES? >> ONE LAST QUESTION FROM STONY BROOK. TOM YOUR EARLIER WORK ADDRESSES IT BUT YOU DIDN'T MENTION IT, IN POPULATIONS THAT ARE EXPOSED TO ARISTOLOCHIC ACID AND STARTING WITH THOSE BELGIAN WOMEN ONLY 10% OR 15% OF THOSE EXPOSED AT THOSE DOSES THAT ARE TAKEN ORALLY ACTUALLY DEVELOPMENT SO MAYBE YOU COMMENT ON THE REASON THAT CONTRIBUTE TO EITHER THE PROTECTION OR THE SENSITIVITY TO IT? >> YEAH, WE DIDN'T--WE DIDN'T REALLY GO INTO THIS, BUT AS ARTHUR SAID ABOUT--IN THE UNFORTUNATE CASE OF THE BELGIAN WOMEN EVEN THOUGH ALMOST ALL OF THEM WERE EFFECTED ONLY 5% WERE SEVERELY EFFECTED TO WHERE THEY LOST THEIR KIDNEYS. MOST OF THEM HAVE RECOVERED. IN THE ENDEMIC REGIONS OF THE BALG ANS IT'S ESTIMATED THAT 5% OF THE POPULATION ARE SENSITIVE AS WELL SO THIS PROBABLY POINTS TO SOME TYPE OF GENERATEDETEC COMPONENT I WOULD BELIEVE TO, YOU KNOW EVEN THOUGH 8 MILLION PEOPLE IN TAIWAN HAVE TAKEN THESE MEDICINE, 8 MILLION PEOPLE HAVE NOT DEVELOPED KIDNEY FAILURE OR CANCERS. SO, YOU KNOW WE BELIEVE THERE IS A GENETIC COMPONENT EITHER IN THE METABOLISM OR IN THE REPAIR OF ADDUCTS OR WHATEVER, OR TRANSPORT OR ANY OF THESE OTHER IMPORTANT ASPECTS. SO WE HAVE LOOKED AT A LOT OF--FROM OUR SEQUENCING LOOKED AT A LOT OF CANDIDATE SNPs AND TYPE THEM AND HUNDREDS OF PEOPLE WE HAVE TO COME ACROSS ANYTHING THAT'S REALLY A SMOKING GUN FOR THE GENETIC FACTORS. >> OKAY. NO MORE QUESTIONS. >> CAN WE CHIME IN JUST 1 MORE TIME. >> GO FOR IT. >> BE SURE TO MUTE IN STONY BROOK, PLEASE? >> SO THE QUESTION THAT I HAD IS IN REGARD TO THE EXTREMELY HIGH MUTATION FREQUENCY THAT YOU'RE SEEING AND IT'S SIM IN TERMS OF RATES AS TO WHAT YOU SEE IN THE MALIGNANT MEL AN OHMSA IN WHICH A LOT OF IMMUNOTHERAPYS WITH THINGS LIKE EP-LIEWM NAB ARE BEING USED FOR THE SUCCESSFUL TREATMENT OF THE MAL8 HOURSINANT MELANOMAS AND I'M WONDER FIGURE THE HIGH FREQUENCY OF MUTATIONS THAT YOU'RE SEEING WITHIN THESE CANCERS WOULD LIMIT ITSELF WELL TO THE IMMUNOTHERAPYS SUCH AS SAY WITH EPI-LUMINAB? >> PLEASE UNMUTE? >> WAS I NOT ON THERE? AM I ON NOW? >> YOU ARE NOW. >> OKAY. THAT WAS A VERY GOOD POINT YOU MADE ABOUT THE HIGH MUTATION RATE AND THAT HAS OCCURRED TO NOT JUST US BUT A LOT OF OTHER GROUPS THAT ARE ADDRESSING THIS. THERE ARE CLINICAL TRIALS IN PROGRESS RIGHT NOW IN TAIWAN LOOKING AT THE EFFICACY OF RENAL THERAPY IN THESE CANCERS AND YOU KNOW 1 COMPONENT WILL--WE'RE INVOLVED IN 1 OF THESE EFFORTS TO SEQUENCE THESE TUMORS AND SEE IF THERE'S A CORRELATION IN JUST THE RAW NUMBER OF MUTATIONS AND THE EFFICACY OF THE TREATMENTS. SO, I CAN'T REALLY SAY ANY CONCLUSIONS WIGHT YET BUT YOU WOULD THINK, YOU KNOW IN OTHER CANCERS THAT'S SHOWN TO BE THE CASE SO I WOULD HOPE THAT YOU KNOW THESE PEOPLE WILL GENERATED FIT IN THE SAME WAY. >> GREAT, THANK YOU VERY MUCH. APPRECIATE IT. >> ARE THERE MORE QUESTIONS? IF NOT I WANT TO THANK YOU VERY MUCH FOR A WONDERFUL TALK.