GOOD AFTERNOON. STEVE ACCEPTD THE INVITATION TO COME SPEND A DAY AT NIH STEPHEN ELLEDGE IS HARVARD PROFESSOR HE'S ALSO AN HHMI INVESTIGATOR HAS BEEN SINCE 1993. AND YOU HAVE SEEN ON THE FLYER, HE WAS BORN IN PARIS. NOT FRANCE. NOT TEXAS. NOT VIRGINIA. BUT ILLINOIS. HE TRAINED AS A CHEMIST, AND THEN MOVED ON TO BIOLOGY, WENT TO MIT. AND WORKED WITH GRANT WALKER, WHERE HE ENGAGED ON DNA, THE FIRST TRANSITION SYNTHESIS POLYMERASE, THEN MOVED ON ACROSS THE COUNTRY TO GO TO STANFORD BIO CHEMISTRY WITH RONALD DAVIS WHERE HE ENGAGED ON THE DNA DAMAGE RESPONSE, A TERM THAT WE ALL USE NOW, THE DDR, WHICH STEVE WAS THE PIONEER FOR THIS PATHWAY WITH THE ATR. HE MOVED TO BAYLOR, 1989, AND HE STAYED THERE FOR 14 YEARS. UNTIL HARVARD MADE A GOOD OFFER TO HIM AND HE WENT TO HARVARD MEDICAL SCHOOL. STEVE IS A MEMBER OF THE NATIONAL ACADEMIA OF SCIENCE, ALSO AN AMERICAN ACADEMIA OF ART AND SCIENCE, AND IN 2004, ELECTED AS A FELL OF THE AMERICAN ACADEMY OF MICROBIOLOGY. HIS LATEST PRIZE WAS IN DECEMBER, 2016, THE BREAK THROUGH PRIZE IN LIFE SCIENCE. IF YOU GO TO HIS WEBSITE YOU WILL SEE WHAT IT ENTAILS, PRETTY PRESTIGIOUS PRIZE AND THE WEBSITE IS BEAUTIFUL. SO I WOULD ENGAGE YOU TO GO THERE. STEVE HAS MOVED ALONG ACROSS HIS CAREER, STARTING FROM VERY DETAILED CHEMICAL ELEMENTS TO DNA DAMAGE, AND DNA RESPONSE, AND THEN ENGAGING IN LARGER DATASET SO IT'S REALLY INSPIRING TO SEE HOW MUCH HE'S MOVED ALONG AND HOW MUCH HE CAN COVER. THANK YOU FOR COMING, LOOK FORWARD TO YOUR TALK. THANK YOU FOR THAT KIND INTRODUCTION. AND THANK YOU FOR THE INVITATION INVITATION. TO TALK ABOUT MY WORK TODAY. SO BASICALLY, I WANT TO TALK ABOUT OUR WORK ON CANCER GENOMICS, UNDERSTANDING HOW ANEUPLOIDY DRIVES CANCER. WE KNOW THAT WHEN WE THINK ABOUT HOW NORMAL CELLS EVOLVE INTO CANCER CELLS WE TYPICALLY THINK OF THE OCCASIONAL DELETION OR AMPLIFICATION. MOSTLY WE FOCUS ON MUTATIONS IN GENES, DRIVER [TECHNICAL DIFFICULTIES]. WHAT I'M GOING TO TALK ABOUT ARE 4 THINGS. FIRST I'LL GIVE YOU AN OVERVIEW OF THE MODELS OF HOW GENE DOSAGE CHANGES, HOW THESE CHANGES DRIVE ANEUPLOIDY PATTERNS AND DRIVE CANCER. I'LL TALK A LITTLE BIT ABOUT NEW EXPERIMENT WE'RE DOING TO LOOK FOR NEW DRIVERS OF CANCER. THESE ARE DRIVERS THAT CAN'T BE IDENTIFIED BY MUTATIONAL SIGNATURES BUT ARE PRESENT IN AMPCANS. THEN I'LL TALK ABOUT THE UNDERLYING GENETIC ARCHITECT OF CASH DRIVERS THAT ARE -- CANCER DRIVERS THAT ARE TISSUE SPECIFIC, WHAT THAT MEANS FOR CANCER GENE IDENTIFICATION, SOMETHING TAIL MUTATION PATTERNS. THEN I'LL PROVIDE EVIDENCE THAT ARGUES THAT ANEUPLOIDY DRIVES TWO HALLMARKS OF CARP, CELL PROLIFERATION AND EVASION OF THE IMMUNE SYSTEM. ANEUPLOIDY IS NOT ONLY A MARKER OF CANCER BUT ACTIVELY PARTICIPATING IN CANCER. SO WE FIRST GOT INTERESTED IN THIS IN 2012. IT CAME THROUGH A VERY SIMPLE SORT OF RNA SCREEN WE DID ON NORMAL EPITHELIAL CELLS. WE ASKED WHAT -- IF YOU KNOCK DOWN THE GENES WHICH GENES -- WHICH MAKES IT GROW SLOWER, AND FASTER? SO THE JEEPS THAT MAKE YOU GROW FASTER WHEN WE KNOCK DOWN THEM, WE CALL THEM [INDISCERNIBLE] GENES. THEY SLOW DOWN THE CELL CYCLE SO THEY'RE NEGATIVE. STOP STANDS FOR SUPPRESSERS OF TUMORIGENESIS OR PROLIFERATION. THINGS THAT MAKE THE CELLS GROW SLOWER WE CALL GOUGINGS. VERY SIMPLE. MOST OF THESE, BECAUSE IT'S A NORMAL CELL LINE, ARE CERTAINLY GENES. SOME ARE ONCOGENES. THE EFFICIENCY THING WE NOTICED, IN THE COLLECTION OF STOP GENES, WE WERE HIGHLY ENRICHED FOR GENES THAT WERE KNOWN TO DRIVE CANCER, KNOWN DO YOU REMEMBER SUPPRESSERS. WE NOT ABOUT MAYBE SOME OF THESE OTHER GENES ARE ACTUALLY ALSO TUMOR SUPPRESSERS IN THIS COLLECTION. SO THAT'S WHERE WE GOT INTERESTED IN THIS. SO WE WENT LOOKING FOR THESE. AT THE TIME, A PAPER HAD BEEN PUBLISHED BY MATT MEYERSON'S LAB THAT MAPPED OUT RECURRING DELETIONS AND AMPLIFICATIONS IN CANCER. WE COMPARED THE GENE SETS TO THOSE, TO LOOK FOR GENES THAT MIGHT BE IN THESE DELETION REGIONS THAT COULD BE FUNCTIONAL DRIVERS OF CANCER. AND THAT LEAD TO A WHOLE ANALYSIS THAT BROUGHT US TO A MODEL, A CANCER GENE ISLAND MODEL. AND WHAT WE FOUND IN FOCAL DELETIONS, THEY WERE ENRICHED FOR THESE STOP GENES. THE GENES THAT WE SHOWED WERE NEGATIVE REGULATORS. THEY WERE PRESENT ABOUT 20% MORE FREQUENTLY THAN YOU WOULD EXPECT RANDOMLY. BUT EVEN MORE IMPORTANTLY, WE FOUND THAT THEY WERE ENRICHED FOR THOSE BUT THEY WERE ACTUALLY AVOIDING THE GO GENES. SO THE FREQUENT FOCAL DELETION AREAS IN CANCER GENOMES ARE ENRICHED FOR STOP GENES AND THEY AVOID ESSENTIAL GENES. THEY AVOID THESE AT THE RATE OF ABOUT 30 TO 35%. AND WE THINK THAT THIS IS A SIGNATURE OF HAPLOINSUFFICIENTCY, BECAUSE THE OTHER COPY OF -- THESE ARE ALL HEMI ZYGOUS DELETIONS. THE OTHER COPY OF THE CHROMOSOME HAS ALL THESE GENES. YOU'RE AVOIDING THE LOSS OF ONE COPY. SO WE HYPOTHESIZED THAT 35% OF ALL HUMAN GENES ARE HAPLOINSUFFICIENT, AND THIS IS IN 2012, SINCE THEN, THERE HAS BEEN A LOT OF SUPPORT FOR THIS BOTH IN THE MOUSE KNOCKOUT CONSORTIUM, BUT ALSO FROM A RECENT SEQUENCING OF 670,000 GENOME -- 60,000 GENOMES WHICH LOOKED AT THE DISTRIBUTION OF LOSS OF FUNCTION ALLELES IN GENES, AND THERE ARE ABOUT 20% OF THOSE -- OF HUMAN GENES HAVE FEWER STOP MUTATIONS THAN YOU WOULD PREDICT. THIS IS A SINGLE COPY ALSO. SO THAT'S ANOTHER SIGNATURE OF HAPLOINSUFFICIENTCY. WE THEN LATER SHOWED IN FOCAL AMPLIICATIONS, YOU SEE THE OPPOSITE. YOU AVOID THE STOP GENES, THE INHIBITOR GENES AND ENRICHED FOR ONCOGENES. AT THIS POINT ESSENTIAL GENES DON'T MATTER, BUT ONCOGENIC DRIVERS DO. SO THIS IS THE FOCAL EVENTS. WHAT ABOUT WHOLE CHROMOSOMES OR ARM EVENTS? AND SO THAT LED US TO DO AN INVESTIGATION OF THESE WHOLE ARMS HERE, AND WE BUILT A MODEL THAT WE CALL THE GENE, CANCER GENE DOSAGE BALANCE MODEL. WE MEASURED THE LIKELIHOOD THAT ANY PARTICULAR ARM OF A CHROMOSOME WOULD BE MORE PRO TUMORIGENIC BASED ON THE DISTRIBUTION OF TUMOR SUPPRESSERS AND ONCOGENES ALONG THE ARMS. AND IF THIS BALANCE, THEN, WOULD MAKE AN ARMOR ANTI-GROWTH AND PRO DELETION OR MORE PRO AMPLIFICATION AND DRIVE THE EUCARYOT. SO WE BUILT A MATHEMATICAL MODEL THAT DID THIS, AND WE WAITED -- WEIGHTED THE TUMOR SUPPRESSER GENES PER ARM, DIVIDED BY THE NUMBER OF GENES PER ARM. THERE IS A CERTAIN TOXICITY, PROTEOTOPIC STRESS THAT WE FIGURED WAS PROPORTION TOOL THE SIZE OF THE CHROMOSOME. SO WE GAVE IT A SCORE FOR THE NUMBER OF IMPOTENCY, AND THEN WE SUBTRACT OUT FOR DELETIONS THE NUMBER OF -- SCORE FOR ONCOGENES AND ESSENTIAL GENES OUT OF THIS. WE GET A COMPOSITE SCORE THAT TELLS YOU HOW TUMOR SUPPRESSER LIKE THAT ARM IS. THEN WE PLOTTED IT AGAINST THE ARM DELETION FREQUENCY. WHAT YOU CAN SEE IS THAT WE GOT A GOOD CORRELATION HERE WITH THE R VALUE OF .75. THIS IS FOR CHROMOSOME ARM LOST. THE SAME IS TRUE FOR WHOLE CHROMOSOMES. THAT SUGGESTS THAT -- SO THE GENES, TUMOR SUPPRESSERS THAT WE'RE WORKING WITH NOW, I FORGOT TO MENTION, THESE ARE NOT FROM THE SHR AND A SCREEN. THESE ARE DERIVED FROM MUTATION OF CANCER GENOMES. SO WE BUILT ANALOGY ALSO TO PREDICT -- ALGORITHM TO PREDICT FROM THE 8,000 GENES AND CONTROLS THAT THE. THE CGA -- TCGA SEQUENCED. THIS ARGUES THE DOSAGE OF THE SAME GENES THAT WHEN MUTATED AT POINT MUTANTS, DRIVE CANCERS. NOW IT'S A CUMULATIVE DOSAGE SHORT OF MODEL. THAT'S THE BACKGROUND. MOST OF THIS IS PUBLISHED SO I WON'T GO INTO IT. NOW I'D LIKE TO TALK ABOUT OUR ATTEMPTS TO IDENTIFY ONCOGENIC DRIVES THAT WERE MISSED BY MUTATIONAL ANALYSIS. SO IDENTIFYING ONCOGENES IS REALLY DIFFICULT. THE WAY THAT YOU DO IT BY SEQUENCE IS LOOK FOR A PATTERN, RECURRING PATTERN LIKE, FOR EXAMPLE, RAS. THERE ARE A SMALLer NUMBER OF WAYS THAT I CAN ACTIVATOR A PREPARE, AS OPPOSED TO INACTIVATE IT. SOME PROTEINS ARE V DIFFICULT TO ACTIVATE BY POINT MUTATIONS. IF YOU INCREASE THE DOSAGE, YOU GET A GAIN OF FUNCTION PHENOTYPE. SO IN ORDER TO LOOK FOR THESE GENES, WE WENT TO DOING MORE GENETIC SCREENS. WHAT WE DID, WE BUILT AN OPEN READING FRAME LIBRARY OF ABOUT 30,000 SEQUENCE VERIFIED ORFs THAT REPRESENT ABOUT 16,000 GENES. AND WE BAR CODED THESE AND PUT THEM UNDER INDUCIBLE PROMOTER. THIS SHOWS YOU WHEN YOU HAVE GFP, IF YOU ADD THE TE.T ANALOG YOU INDUCE THE EXPRESSION. WE PUT THEM INTO NORMAL CELLS. WE PUT THEM INTO HUMAN MAMMARY EPITHELIAL CELLS. THESE ARE EMOTERALIZED. PANCREAS OR FIBROBLASTS. THEM WE ADD DOX OR DON'T. WE LET THEM GROW FOR TEN GENERATIONS, LOOK TO SEE WHICH GENES ENRICH, WHICH GENES MAKE THE CELLS NOT PROLIFERATE. SO THIS IS WHAT THE DATA ACTUALLY LOOKS LIKE. YOU CAN SEE THAT WE GET ENRICHMENT OF SOME GENES, WE CALL THESE GO GENES BECAUSE PROMOTING GROWTH. AND WE SEE THE DROPOUT OF A LOT OF GENES, WE CALL THESE STOP GENES. WHEN YOU LOOK AT THE TYPES OF GENES THAT ARE DRIVING THE CELL CYCLE IN PROLIFERATION, THERE ARE A LOT OF FAMILIAR FACES LIKE MIC, CDK6, RECEPTOR TYROSINE KINASES, THINGS LIKE THAT. THERE A LOT OF GENES WE NEVER HEARD OF BEFORE THAT. WAS ONE OF THE BIG SURPRISES. AND THINGS THAT DROP OUT, A LOT OF THINGS YOU MIGHT EXPECT, THE CDK MIGHT BE SHOWN ON THE LOWER RIGHT BUT ALSO INTERFERON JONES, SLOWS CELLS, MAKES THEM SICK. WHEN YOU MAP THE POSITIONS OF THESE GO GENES ON TO A MAP OF CHROMOSOMES, WHERE THE AMPCANS ARE SHOWN, YOU CAN SEE THAT THAT WE FILL IN A LOT OF GAPS. WE FOUND ALMOST 150 PRO PRO LEGISLATERATION GENES IN RECURRING AMP PLICANS. AND THE RECURRING AMP PLICANS MAPPED HERE ARE EITHER PAN CANCER, IN A LOT OF CANCERS, OR SPECIFICALLY IN BREAST OR PANCREATIC CANCERS. WE DIDN'T DO SARCOMAS. WE DON'T KNOW HOW MANY OF THE DRIVERS ARE FUNCTIONAL BUT WE FOUND 50% OR GREATER ENRICHMENT FOR THESE GENES IN THESE REGIONS THAN YOU WOULD EXPECT RANDOMLY. SO WE THINK WE HAVE SOME DRIVES. NOW I WANT TO TALK ABOUT HOW THESE DRIVER BEHAVE IN DIFFERENT CELL TYPES. THERE WE FOUND SOMETHING SURPRISING TO US. AS I SAID WE DID 3 NORMAL CELL LINES, SO WHAT WE FOUND WERE THAT THE TWO EPITHELIAL LINES, THE BREAST AND PANCREATIC LINES, WERE -- HAD THE MOST SIMILAR PATTERN OF GENES RELATIVE TO FIBROBLASTS. SO THAT MAKES SENSE AT SOME LEVEL. AND WHEN YOU LOOK AT THE OVERLAP BETWEEN THEM WHAT YOU CAN SEE HERE ON THE RIGHT, IF YOU FOCUS ON THE STOP GENES, THERE IS A LOT OF OVERLAP. DEPENDING ON WHICH SIDE YOU LOOK AT IT'S BETWEEN 40 AND 63% OVERLAP. AND THERE ARE A LOT OF GENES THAT MAKE CELLS GROW SLOWER. THIS IS ALL THE SAME STATISTICAL CUT OFF. THERE ARE A LOT OF WAYS TO TURN OFF CELL PROLIFERATION, MAKE THE NOT WORK WELL. THE BIG SURPRISED WERE THE GENES THAT MADE CELLS PROLIFERATE. FROM THERE WE GOT A VERY SMALL OVERNIGHT LAP. WE THOUGHT -- OVERLAP. WE ONLY GOT ABOUT A 10% OVERLAP BETWEEN THESE GENES. THAT WAS REALLY SURPRISING TIES. NOT ONLY DID WE NOT SEE THAT MUCH OVERLAP, ACTUALLY, THE REVERSE OVERLAPS BETWEEN THE GO GENES FOR H MIX AND -- VERSES THE STOP GENES FOR HP AND E, THEY HAD ALREADY THE SAME OVERLAP, WHICH IS SHOWN ON THE LEFT. HERE ARE THE GO GENES. IT'S ALMOST THE SAME PERCENTAGE. A LITTLE BIT LESS THAN WHEN YOU COMPARE THE GO VERSES GO GENES. THE SAME IS TRUE OVER HERE FOR THE OPS THE COMPARISON. SO -- OPPOSITE COMPARISON. IF YOU LOOK AT ALL 3 OF THE CELL TYPES THAT WE LOOKED AT AND WE HAVE 2 DIFFERENT LIBRARIES. THIS IS ONLY ONE OF THE LIBRARIES. SO THE NUMBERS ARE A LITTLE SMALL. WHAT YOU CAN SEE IS THAT THE OVERLAP BETWEEN ALL 3 OF THESE CELL LINES, THEY'RE ONLY 3 GENES THAT ARE IN COMMON. SO THIS SUGGESTED TO US THAT THESE CELLS ARE REALLY WIRED DIFFERENTLY. THAT THEY'RE GOING TO RESPOND VERY DIFFERENTLY TO DIFFERENT TYPES OF GROWTH SIGNALING. AND SO THAT WAS REALLY THE BIG TAKE HOME FROM THAT. THE OTHER THING THAT WE FOUND THAT WAS INTERESTING RELATED TO THE KINDS OF GENES IN THESE LIBRARIES THAT ARE KNOWN TO BE MUTATED IN THESE TUMORS, SO WE LOOKED AT COMMON ONCOGENES AND TUMOR SUPPRESSERS AND HOW THEY BEHAVE. IN THIS GRAPH, WE JUST HAVE A -- ON THIS, WE HAVE THE HP AND E PANCREATIC CELLS. IT GIVES THE P VALUE OF WHETHER THEY DROP OUT OR ENRICHED. THE SAME THING IS TRUE FOR THE MAMMARY EPITHELIAL CELLS, IF THEY'RE ENRICHED, THEY GO UP HERE. IF THEY DROP OUT THEY GO DOWN HERE. YOU CAN SEE THAT THERE ARE A LOT OF COMMON DRIVERS THAT DRIVE CANCER. ACT LIKE GO GENES. AND THERE ARE GENES THAT ARE KNOWN TO INHIBIT PROLIFERATION IRON COMMON. NOW, IF YOU LOOK SPECIFICALLY AT BREAST, WHAT YOU SEE IS THAT THE ONCOGENES THAT DRIVE BREAST CANCER, P-GP RECEPTOR SCORE WELL, THEY DID VIRTUALLY NOTHING IN PANCREATIC CELLS. THESE 3 TUMOR SUPPRESSERS IN BREAST, WHEN YOU OVER-PRODUCE THEM THEY DROP OUT OFF THESE, BUT THEY HAVE NO EFFECT AT ALL ON HPNE CELLS. THE SAME IS TRUE FOR THE TUMOR SUM PRESSESERS THAT ARE SPECIFIC TO PANCREATIC CANCER. YOU CAN SEE THE ONCOGENES, THEY STIMULATE THESE CELLS TO PROLIFERATE BY THEY DO NOTHING IN BREAST CANCER. THEY'RE NEVER MUTATED IN BREAST CANCER, LIKEWISE, TUMOR SUPPRESSER, TG.F. BETAS RECEPTOR DOES NOTHING IN BREAST BUT DROPS OUT OF THESE PANCREATIC CELLS. WE CAN LOOK AT THE GROUP OF GENES THAT FUNCTION IN THESE DIFFERENT CELL TYPES AND PLOT THEM BASED ON EITHER THINGS THAT WORK IN BOTH OF THESE CELL LINES, THE MAMMARY AND THE PANCREATIC CELL LINES. THESE ARE BASIC CDK INHIBITERS AND MK, THE WNT PATHWAY. YOU START TO SEE GROUPS OF GENES THAT FUNCTION ONLY IN ONE CELL TYPE. KNEES WERE GENE ONTOLOGY CATEGORIES WE FOUND. ONE ARE GENES SPECIFICALLY INVOLVED IN PANCREATIC BETAS CELL DEVELOPMENT, SPECIFICALLY SHOWED UP IN THE PANCREATIC CELLS. ALSO SETS OF PANCREAS SPECIFIC TRANSCRIPTION FACTORS. AND NUCLEAR RECEPTORS. IN BREAST, WE SEE PROST NOWED LIGAND RECEPTORS WHAT YOU MIGHT EXPECT, BUT ALSO BREAST SPECIFIC TRANSCRIPTION FACTORS. I WANT TO POINT OUT IN PARTICULAR A GROUP OF BREAST SPECIFIC PROTEINS CALLED KERTEN ASSOCIATED PROTEINS. AND THESE ARE ACTUALLY ALSO -- NORMALLY EXPRESSED IN THE SKIN. THAT CROSS LINK KER ATTENS. THERE IS TWO FAMILIES, ONE IS VERY SISTINE RICH. THESE GENES, HOW THEY BEHAVED. CERTAIN FAMILIES, ALL THE MEMBERS OF THESE FAMILIES PROLIFERATE AND OTHERS DON'T AT ALL. NOW, IF YOU LOOK AND SAY HOW DO THEY BEHAVE, RELATIVE TO OTHER GENE SETS, WELL, IF YOU COMPARE IT IN MOMRY CELLS, OLFACTOR RECEPTORS ARE NEUTRAL. VIRTUALLY EVERY ONE OF THESE ARE POSITIVE OR HAVE NO EFFECT AT ALL. IF YOU LOOK HOW THEY BEHAVE IN THE PANCREATIC CELLS OR FIBROBLASTS, THEY BASICALLY HAVE A NEUTRAL EFFECT. SO THEY'RE REALLY SPECIFIC FOR STIMULATING THESE NORMAL BREAST CELLS. WE WERE INTERESTED IN WHETHER OR NOT THEY MIGHT BE OVER-EXPRESSED IN CANCER IN GENERAL. WE DID A COMPARISON OF THE TWO DIFFERENT TYPES OF THESE, NEUTRALS AND THE GO GENES, AND YOU CAN SEE THAT FOR THE NEUTRAL GENES, IF YOU LOOK IN THE NORMAL TISSUE OR THE TUMOR TISSUE, THERE IS VIRTUALLY NO DIFFERENCE. BY FOR THE GO GENES, YOU OFTEN SEE WHEREVER THESE ASSTRICTS ARE, THAT THEIR EXPRESSION IS ENRICHED IN THE TUMORS FROM THESE DIFFERENT TUMOR TYPES. THEY APPEAR TO BE SOME POTENTIAL, TO BE EXPRESSED HIGHLY IN TUMORS AND MAY HELP DRIVE THESE TUMORS, AT LEAST POTENTIALLY TO THIS CORRELATION. SO I THINK 7 OF THE 11 TUMORS THAT WE LOOKED AT HEAR STATISTICALLY OVER-EXPRESSED THESE IN TUMORS SPECIFICALLY. WE WANTED TO KNOW HOW THESE TUMOR SPECIFIC DRIVERS MIGHT WORK, SO WE TOOK CELLS, THESE ARE NOW HMK CELLS. WE OVERNIGHT PRODUCED THEM AND LOOKED AT THEIR PROFILES TO SEE HOW THEY BEHAVE. AND THIS IS JUST A PRINCIPLE COMPONENTS ANALYSIS SHOWN OVER HERE USING THE TRANSCRIPTOME DATA AND WHAT YOU CAN SEE IS THAT THIS IS EV, EMPTY VECTOR. AND DETYPE CYCLINS AND KTRAPs ARE LOCALIZED TOGETHER, FARTHER AWAY FROM LOSS OF FUNCTION OF P53, SRC OR MK EXPRESSION. THESE LOOK MORE SIMILAR IF YOU LOOK AT THE CLUSTERING OF GENE EXPRESSION. YOU CAN SEE THAT THE KTRPs AND CYCLIN Ds HAVE A SIMILAR SIGNATUE. THEY OVEREXPRESS CELL CYCLE GENES AND PROTEIN SYNTHESIS GENES. THIS SET IS ALSO COULD BE SERVED WITH MK -- MYC. THEY'RE QUITE DIFFERENT FROM THESE OTHER CANCERS. SO YOU CAN SEE THEY CLUSTER TOGETHER. CYCLIN D AND KRTAPs LOOK VERY SIMILAR. WHAT WE KNOW HOW CYCLIN D DRIVES THE CELL CYCLE, IF YOU LOOK DOWN HERE, PSYCHENS, ACTIVATED TUMOR SUPPRESSER CALLED RB. RB ACTIVATES IN THIS COMPLEX A TRANSCRIPTION FACTOR CALLED E2F THAT AFTER CYCLIN D PHOSPHORYLATES IT, IT ACTIVATED THE G1 TO S TRANSITION. AND SO WE LOOKED AT E2F1 RNA LEVELS. YOU CAN SEE THEY INCREASE WHEN YOU OVEREXPRESS KTRAP BY OVER TWO-FOLD. THIS IS P VALUE HERE. QUITE HIGH FOR THIS. SO WE THINK THAT PART OF THIS SIGNATURE MAY BE BASE KTRAPs ARE DRIVING ENTRY INTO THE CELL CYCLE IN A WAY SIMILAR TO D TYPE CYCLINS. THIS IS NEW DATA SO WE HAVEN'T DONE THE ANALYSIS AND LOOK AT THIS IN MORE DETAIL. WE THINK THERE IS PROBABLY A LINK HERE. THIS LINK SHOULD BE SPECIFIC TO BREAST CELLS. WE DON'T UNDERSTAND THAT, BUT WE THINK IT'S VERY LIKELY TO BE THE CASE. SO THE OTHER THING THAT WE STARTED LOOKING AT IS HOW THESE GENES BEHAVED IN AMPLICONS IN DIFFERENT TYPES OF CANCER. SO IF YOU LOOK AT THE GO GENES THAT WERE FOUND IN THE HMEC SCREEN OR PANCREATIC CELL SCREEN THEY'RE BOTH ENRICHED AS A PERCENTILE IN THE PAN CANCER AMPLICONS, 50-60% MORE THAN YOU WOULD EXPECT. IF YOU, THEN, LOOK IN AMP PLI THE SAME IS TRIIN THE STOP GENES. IN THE PAN CANCER, YOU SEE MORE OF THESE STOP GENES AND RECURING DELETIONS. THE HP AND Es LOOK A LOT BETTER FOR WHATEVER REASON IN THE PAN CANCER. BUT YOU CAN SEE IF YOU NOW LOOK AT DELETIONS SPECIFICALLY IN THE BREAST, THE STOP GENES ARE EVEN MORE ENRICHED. IF THEY WERE FOUND IN HMYCs, YOU SEE IF THEY WERE FOUND IN HPNEs. THE SAME IS TRUE IN PANCREATIC TUMORS, NOW THE HPNEs ARE ENRICHED. SO WE ALSO ASKED THE QUESTION, NOW, SO THESE ARE DIFFERENT CELL TYPES. IF YOU LOOK AT -- IN TUMOR TYPES, IF YOU LOOK AT TUMORS THAT MORE RELATED TO, SAY, BREAST TUMORS OR MORE RELATED TO PANCREATIC CANCERS, DOES THIS PATTERN HOLD? AND SO WE LOOKED FIRST AT OVARIAN AND UTERINE CANCERS THAT ARE MORE SIMILAR TO BREAST CANCERS BECAUSE THEY'RE CANCERS OF THE FEMALE REPRODUCTIVE SYSTEM. WHAT YOU CAN SEE IS THAT THE GO GENES NOW FROM THE HMYC SCREENS ARE MORE ENRICHED AND OVARIAN AMPLICANS THAN ARE THOSE IDENTIFIED IN THE PANCREATIC SCREEN. THE SAME IS TRUE FOR THE AMPLICONS FOUND IN UTERINE CANCER. THE GENES THAT MAKE THE BREAST CELLS GROW BETTER ARE MORE ENRICHED THAN THOSE THAT -- IN THOSE THAN THE GENES THAT MAKE PANCREATIC CELLS GROW BETTER. IF YOU NOW SWITCH AND LOOK AT TUMOR TYPES THAT ARE MORE RELATED TO PANCREATIC CANCER, NOW YOU GET THE OPPOSITE, THAT THE BREAST GO GENES DON'T DO MUCH AT ALL HERE. STOMACH OR COLORECTAL CANCER. BUT THE GO GENES FOR THE PANCREATIC CELLS NOW ARE ENRICHED. SO THIS SUGGESTS THAT THE AMPLICONS THAT YOU SEE -- SO IF YOU LOOK AT AMPLICONS IN DIFFERENT TUMOR TYPES, YOU SEE SOME THAT ARE CONSERVED ACROSS ALL TUMORS, THESE ARE THE PAN CANCER AMPLICONS. AND THEN YOU SEE SOME THAT ARE SPECIFIC TO DIFFERENT TYPES OF CANCER. AND SO ONE OF THE ARGUMENTS ABOUT HOW THESE THINGS WORK IS THAT THEY -- THE ENVIRONMENT IS DIFFERENT. YOU HAVE A HIGHER FREQUENCY IN CERTAIN REGIONS BASED ON THE STRUCTURE OF THE GENOME, DIFFERENT FROM CELL TYPE TO CELL TYPE. THE ALTERNATIVE ARGUMENT IS THAT DIFFERENT GENES ARE BEING SELECTED FOR ONCE THESE EVENTS OCCUR, THEY OCCUR AT SOMEWHAT EVEN FREQUENCY. SELECTION ACTS ON IT. AND IF THAT'S THE CASE, THEN IF THESE WIRED -- IN TERMS OF WHAT GENES THEY RESPOND TO, YOU CAN EXPECT THAT THEIR APPLICANS WOULD BE MORE RELATED IF THE TISSUES WERE RELATED. WE DID -- WE LOOKED AT RECURRING AMPLIFICATIONS AND DELETIONS ACROSS A NUMBER OF DIFFERENT TUMOR TYPES, AND THEN WE DID AN UNSUPERVISED NON -- UNSUPERVISED HIERARCHICAL CLUSTERING. WHAT YOU CAN SEE IS THAT THE AMPLICONS PREDICT THE RELATEDNESS OF THE TISSUES DURING DEPARTMENT, SO THE BREAST TISSUES CLUSTER WITH THE UTERINE AND OVARIAN CANCERS BASED ON THEIR AMPLICONS, AND THE PANCREATIC CANCERS AMPLICONS CLUSTER WITH STOMACH AND COLORECTAL. WE ALSO SEE MELANOMA AND GLIOMAS AND GEOBLASTOMA CLUSTERED TOGETHER, ALL DERIVED FROM THE [INDISCERNIBLE]. SO BASICALLY WHAT THIS ARGUES IS THAT THE WIRING OF ALL THESE DIFFERENT TISSUES DETERMINES WHAT SETS OF GENES ARE ABLE TO INFLUENCE THEIR PROLIFERATION, AND TUMOR GENESIS. AND THAT THESE ARE DRIVING THE APPARENTS OF AMPLIFICATION AND DELETION THAT ARE SPECIFIC TO DIFFERENT TUMOR TYPES. SO NOW IN THE LAST FEW MINUTES OF THIS TALK, I'D LIKE TO TALK ABOUT HOW ANEUPLOIDY ACTUALLY IS DRIVING CANCER. AND WHAT THE IMPACT OF ANEUPLOIDY IS ON CANCER. WE TALKED ABOUT THE DIFFERENT GENES THAT ARE EFFECTED DURING AMPLIFICATION AND DELETION OF CHROMOSOMES AND CHROMOSOME ARMS OR FOCAL EVENTS, WHICH ARE LESS THAN THE GENOMESOME ARM. BUT HOW THAT ACTUALLY WORKS, WHAT IT ACTUALLY DOES TO PROMOTE CANCER IS WHAT WE'RE INTERESTED IN. SO ONCE WE STARTED GIVING SCORES TO ANEUPLOIDY AND TRYING TO PREDICT WHICH CHROMOSOME ARMS AND -- WERE DELETED OR AMPLIFIED IN CANCERS, WE REALIZED WE COULD ACTUALLY DEVELOP A SCORING MAY TRICKS TO ACTUALLY GIVE A PARTICULAR TUMOR AND ANEUPLOIDY LIKE SCORE BASED ON THE NUMBER OF CHROMOSOME ALTERATIONS, CHROMOSOME ARM ALTERATIONS AND FOCAL EVENTS. SO WE GUILT BUILT ANALOGY ALSO OR MATHEMATICAL -- ALGORITHM OR MATHEMATICAL MODEL TO GIVE A SCORE FOR SOMATIC COPY ALTERATIONS FOR EVERY TUMOR. AND THIS IS JUST A HEAT MAP TO SHOW YOU THAT CERTAIN CHROMOSOME ARMS ACROSS ALL CANCERS ARE RECURRING OR CERTAIN ARMS LIKE TO BE LOST. A HEAT MAP, OR GAINED SHOWN IN GREEN. SO WE DID THIS AND THIS ALLOWED US TO, THEN, SEPARATE TUMORS BASED ON THEIR ANEUPLOIDY SCORE. WE TOOK THE TOP 30% OF MOST ANEUPLOIDY AT THIS TWOS AND THE BOTTOM 30, THE LEAST TUMORS A, AND WE LOOKED AT THE TRANSCRIPTOMES OF THOSE TUMORS. THEN WE STARTED TO LOOK FOR SETS THAT WERE ENRICHED IN TUMORS WITH HIGH ANEUPLOIDY. AND SO THIS IS A GENE SET ENRICHMENT ANALYSIS CALLED GSEA. FOR THOSE NOT FAMILIAR WITH IT, THE WAY THIS WORKS IS IF YOU LOOK AT THIS HEAT MAP DOWN HERE, HIGH TO LOW, WE CAN PAIR EVERY GENE FOR THE DIFFERENCE BETWEEN HIGH ANEUPLOIDY AND LOW, AND THEN WE MOTE THE RATIO FROM THE HIGHEST EXPRESSING, DIFFERENTIALLY EXPRESSING, THE HIGH TO THE LEAST, SO THEY'RE MOST EXPRESSED IN LOW ANAPY. AND THAT'S FOR -- ANEUPLOIDY, AN THAT'S FOR ALL THE GENES IN THE WHOLE TRANSCRIPTOME. YOU TAKE GENE SETS AND MAP THEM. IF YOU TAKE A NEUTRAL GENE SET AND MAP IT ON TO EACH OF THESE BLACK BARS IS A GENE, YOU GET A -- IF IT'S A RANDOM GENE SET OR UNINFORMATIVE GENE SET, IT HAS AN EVEN DISTRIBUTION AND YOU CAN BASICALLY GET A STRAIGHT LINE ACROSS. IN THIS CASE, WE LOOKED AT THE DNA REPLICATION GENES. WHAT YOU CAN SEE IS THAT THEY'RE MOST HIGHLY EXPRESSED IN THE HIGH ANEUPLOIDY TUMORS. THIS IS TRUE FOR CELL CYCLE GENES, DNA REPLICATION, AND THESE ARE ALL CELL SIGNINGAL REGULATED GENES TRANSCRIPTS. SO WHAT THAT ARGUES IS THAT HIGH ANEUPLOIDY TUMORS ARE MORE HIGHLY MITOTIC. AND THEY GIVE A ENRICHMENT SCORE FOR THIS, ALL THAT, BUT BASICALLY, YOU CAN JUST SEE BY EYE THAT THEY'RE ALL PILING UP TO ONE SIDE. AND SO THIS MAKES SENSE BECAUSE A LOT OF THE GENES THAT ARE DRIVING ANEUPLOIDY ARE THESE GENES THAT EFFECT THE CELL CYCLE. SO -- AND THIS HAD BEEN SEEN PREVIOUSLY IN DATA IN CELL LINES, BUT THIS IS, I THINK, THE FIRST TIME THAT ANYONE HAD SEEN IT IN TUMOR CELL TYPES. SO THAT MAKES SENSE, HIGH ANEUPLOIDY SELECTED FOR, SELECTING FOR A HIGHER FREQUENCY OF MITOSIS, AND CELL CYCLE. THAT DRIVES CANCER. BUT THE OTHER THING THAT WE FOUND THAT WAS SURPRISING THAT WASN'T KNOWN PREVIOUSLY WAS THAT WE FOUND THAT THERE WAS A GENE SET THAT WAS ACTUALLY DEPLETED IN HIGH ANEUPLOIDY TUMORS. AND THESE ARE INVOLVED IN THE IMMUNE CELL MARKERS. NOW, TUMORS CELLS DON'T EXPRESS THESE IMMUNE CELLING MARKERS, OF COURSE. THEY'RE SPECIFIC TO IMMUNE CELLS. WHEN YOU TAKE THE WHOLE TUMOR AND SEQUENCE IT, YOU TAKE EVERYTHING FROM THE TUMOR. THAT INCLUDES CELLS THAT INFILTRATE THE TOMB, IF COLLUDING THE IMMUNE CELLS. IF YOU LOOK AT THE PLOTS HERE FROM GSEA, YOU CAN SEE ALL THESE GENE SETS FOR EITHER CD8 AND NATURAL KILLER CELLS, INTERFERON GAMMA SIGNALING, THEY'RE ALL OVER ON THE RIGHT. SO THAT -- WHAT THAT SAYS IS THAT LOWE TUMORS HAVE A HIGHER IMMUNE INFILTRATE THAN HIGH TUMORS. SO WHAT WAS PREVIOUSLY KNOWN ABOUT THE IMMUNE TUMOR WAS A PAPER BY [INDISCERNIBLE] LAB WHO SHOWED THAT THE NUMBER OF MUTATIONS OR NEO EPITOPES IN A PARTICULAR TUMOR POSITIVELY CORRELATED WITH IMMUNE INFILTRATION. THAT MAKES SENSE, THE MORE MUTATED YOU ARE, THE MORE LIKELY YOU'LL BE NOTICED BY THE IMMUNE SYSTEM. FOR 3 TUMORS HERE, THESE ARE ALL DIFFERENT TUMOR TYPES. THESE ARE COLORECTAL CANCERS, LUNG ADENOMAS, AND UTERINE ENDOMETRIAL CANCERS. THERE IS A POSITIVE CORRELATION. NOW, THE P VALUE -- THE CORRELATION COEFFICIENT IS PRETTY WEAK, ABOUT .2. THE P VALUES ARE BARELY SIGNIFICANT. TEN TO THE MINUS 2, TEN TO THE MINUS 3. BUT THERE IS THIS POSITIVE CORRELATION. BUT -- JUST PAY ATTENTION TO THESE 3 WITH THE ASTERISKS, BECAUSE I'M GOING TO SHOW YOU IN CONTRAST WHAT IT LOOKS LIKE FOR THIS CORRELATION WITH ANEUPLOIDY. AND THAT'S SHOWN HERE. SO IF YOU DO THIS CORRELATION NOW, INSTEAD OF THE MUTATION NUMBER YOU DO IT WITH THE ANEUPLOIDY SCORES. YOU SEE YOU GET A NEGATIVE CORRELATION. THE HIGHER THE ANEUPLOIDY SCORE, THE LOWER THE IMMUNE INFILL TRANSPLANT SCORE. FOR THESE 3. SO INSTEAD OF .2 WE HAVE CORRELATIONS OF ALMOST .4 AND P VALUES BETWEEN 10 TO THE MINUS 6 AND 10 TO THE MINUS 9. SOME OF THESE ARE EVEN STRONGER, HEAD, NECK, THE CORRELATION COEFFICIENT IS .45. THE P VALUE IS TEN TO THE MINUS 16. THE ONLY TWO TUMOR TYPES THAT DON'T SHOW THIS ARE BRAIN CANCERS. THIS IS LOW GRADE GLIOMA. AND GLIOBLASTOMA MULTI FORM, ACTUALLY HAS NEGATIVE CORRELATION THERE. SO -- OR POSITIVE CORRELATION OVER HERE FOR THE LOW GRADE GLEOOMAS. SO FOR ALL THE EPITHELIAL CANCERS, WE SEE A STRONG CORRELATION. THIS IS JUST ANOTHER WAY TO LOOK AT IT. AND SO WHILE I'M PLOTTING HERE IT'S A HEAT HAPPEN OF THE FALSE DISCOVERY RATES FOR THESE. SO .01 IS VERY LOWE FALSE DISCOVERY RATE. YOU CAN SEE JUST BY LOOKING AT THE COLORS THAT THE BRAIN CANCERS DON'T FOLLOW THESE RULES. BUT ALL THE OTHER CANCERS DO. OVARIAN, STOMACH ADENOMA, MELANOMA, KIDNEY CANCER, BREAST CANCER, COLORECTAL CANCERS. THESE ARE THE GENE ONTOLOGY CATEGORIES AND THEY'RE KIND OF HARD TO UNDERSTAND. LET ME BREAK IT DOWN IN TERMS OF CELL TYPES. WE BUILT UP A CELL TYPE SPECIFIC SIGNATURE. WHAT YOU CAN SEE IS THAT THE CD8T CELL, EFFECTER CELLS, ARE ARE -- SHOW A SIGNIFICANTLY REACCUSED LEVELENED MAY APANNIE TUMORS AS DO B CELLS, NATURAL KILLER CELLS, AND ALSO T REGULATORY CELLS. T REGULATORY CELLS ARE NEGATIVE REGULATORS, SO YOU MIGHT THINK THAT GOES IN THE WRONG DIRECTION. I WANT TO POINT OUT ALTHOUGH THEY'RE DEPLETED, THEY'RE NOT DEPLETED NEARLY AS WELL. YOU CAN SIGH THAT FALSE DISCOVERY RATE IS MUCH HIGHER FOR THESE. BUT THE RATIO OF T REGULARS AND CD8 CELLS TO T REGULARS IS VERY SIGNIFICANT. SO WHAT'S -- WHAT LITTLE IMMUNE ENVIRONMENT IS LEFT IN THESE TUMORS WITH HIGH ANEUPLOIDY, IT'S EVEN MORE ANTI-IMMUNOGENIC. SO NOW THAT WE'VE IDENTIFIED THESE TWO DIFFERENT GENE SETS, THESE SELL CYCLE GENE SETS AND IMMUNE INFILTRATE GENE SETS WE CAN ASK MORE SPECIFIC QUESTIONS LIKE WHAT ASPECTS OF ANEUPLOIDY ACTUALLY DRIVE THESE TWO DIFFERENT RESPONSES? SO THE WAY THAT YOU CAN DO THIS IS THAT WE'RE TAKING ADVANTAGE OF A MATHEMATICAL METHOD CALLED LASSO, WHICH IS SORT OF A MACHINE LEARNING METHOD WHERE YOU CAN FEED PARAMETERS INTO IT AND ASK THEM TO WEIGH THE PARAMETERS AND SAY HOW BEST CAN YOU WEIGHT THIS PROGRAMMER TO PREDICT THIS PARTICULAR OUTCOME, LIKE LOW ANEUPLOIDY OR HIGH CELL CYCLE GENE EXPRESSION. YOU FEED IN DIFFERENT PROGRAMMERS, AND THEN YOU ASK FOR IT TO WEIGHT THOSE. IT GIVES YOU A BETA COEFFICIENT WHICH TELLS YOU HOW MUCH TO WEIGHT IT. A LOT OF TIMES YOU PUT IN PARAMETERS, IT WILL JUST GIVE YOU 0. THEY DON'T PREDICT ANYTHING AT ALL. WHEN THEY HAVE A POSITIVE PREDICTION, IT WILL GIVE YOU A BETA PARAMETER. THAT CAN BE POSITIVE OR NEGATIVE DEPENDING ON WHETHER OR NOT IT IS PREDICTIVE OR ANTI-PREDICTIVE. AND SO WHAT WE -- THE PARAMETERS WE PUT INTO THIS PARTICULAR ANALYSIS WERE FOLKLING -- FOCAL SOMATIC COPY NUMBER ALTERATIONS OR WHOLE CHROMOSOME AND ARM ALTERATIONS. IF I LOOK AT THE TOP HERE, THE CELL CYCLE SIGNATURE, IT GAVE BETA COEFFICIENTS FOR BOTH OF THESE THAT WERE POSITIVE, AND THE FOCAL COEFFICIENT IS HIGHER THAN THE COEFFICIENT FOR THE ARM. THAT MEANS THEY BOTH -- THEY'RE BOTH POSITIVE. FOCAL EVENTS ACTUALLY HAVE A MORE POTENT ABILITY TO PREDICT THIS IMMUNE SIGNATURE. AND THEN WHOLE CHROMOSOMES AND ARMS. THAT MAKES A CERTAIN AMOUNT OF SENSE BECAUSE WHEN YOU'RE -- WHEN YOU THINK ABOUT A FOCAL RECURRING FOCAL EVENT, YOU'RE BASICALLY PICKING OUT THE SORT OF SWEET SPOT OF THE GENOME THAT IS MOST ENRICHED FOR THE GENES YOU'RE INTERESTED IN AND AVOIDING THE GENES YOU DON'T WANT THERE. AND IF YOU, THEN, TAKE THE WHOLE CHROMOSOME ARM YOU'RE SORT OF TAKING THE GOOD WITH THE BAD. SO IT SHOULD BE LESS POTENT OF AN EVENT, AND THEREFORE, BE SELECTED LEST EFFICIENTLY. SO -- AND BE LESS PREDICTIVE. THAT'S EXACTLY WHAT YOU SEE HERE. NOW, IF YOU LOOK AT THE IMMUNE SIGNATURE, WE GET SOMETHING THAT WAS REALLY UNEXPECTED. WHAT YOU CAN SEE HERE IS THAT NOW THE FOCAL EVENTS HAVE VERY LITTLE PREDICTIVE VALUE. BUT THE WHOLE CHROMOSOME AND WHOLE ARM EVENTS ARE VERY POTENT. SO DIFFERENT ASPECTS OF ANEUPLOIDY ARE PREDICTING THE TWO DIFFERENT SIGNATURES. AND IF YOU LOOK AT -- INSTEAD OF FOR PAN CANCER, IF YOU LOOK ACROSS ALL CANCERS, WHAT YOU CAN SEE IS THAT YOU PRETTY MUCH SEE THAT THE FOCAL EVENTS ARE THE STRONGEST UP HERE BUT THERE IS A LOT OF -- ON THIS HEAT MAP, THERE IS A LOT OF ARM EVENTS, WEIGHTING OF ARM EVENTS. BUT FOR AN IMMUNE SIGNATURE IT'S ALMOST ALL WHOLE CHROMOSOME AND WHOLE ARM EVENTS. THE EXCEPTIONS ARE GLIOBLASTOMA, THAT GOES THE WRONG DIRECTION. ALL THE EPITHELIAL CANCERS LOOK THIS WAY. SO I THINK WHAT THIS ARGUES IS THAT THERE ARE FUNDAMENTALLY DIFFERENT MECHANISMS THAT DRIVING THESE TWO DIFFERENT SIGNATURES. THE FIRST ONE IS DRIVEN BY FOCAL EVENTS AND GENES, PARTICULARLY SETS OF GENES. BUT THE SECOND ONE LIKELY IS A PROPERTY OF THE WHOLE CHROMOSOME. AND NOT PARTICULAR GENE SETS. I'M NOT SAYING THAT GENES DON'T DRIVE IT. WE KNOW THAT'S NOT TRUE. GENES DRIVE IMMUNE EVASION, NO QUESTION ABOUT IT. BUT WE THINK THAT WHOLE CHROMOSOMES DO, TOO. AND THAT THAT'S PROBABLY SOME PROPERTY THAT'S UNIQUE TO THE PROTEOTOXIC STRESS THAT IS DUE TO THE IMBALANCE OF COMPLEXES ACROSS THE PROTEOME, BECAUSE WHEN YOU MAKE AN ENTIRE CHROMOSOME, A LOT OF THE GENES ARE MADE AT THE WRONG -- PROTEINS ARE MADE AT THE WRONG LEVELS. THEY END UP BEING TURNED OVER AND THAT GENERATES PROTEOTOXIC STRESS. WE FED INTO THE LASSO A LOT OFF OTHER PARAMETERS. THIS PRETTY MUCH HELD FOR THE IMMUNE INFILTRATE. WE PUT IN THE NUMBER OF MUTATIONS AND VARIOUS NUMBER OF OTHER PARAMETERS, TUMOR STAGE, AGE, GENDER. WHAT WE FOUND, AGAIN, WAS THAT THE MAIN PREDICTOR IS CHROMOSOME ARMS, OCCASIONALLY YOU'D SEE FOCAL EVENTS. IT'S ALWAYS LOWER MAGNITUDE. AND ALSO THE NUMBER OF MUTATIONS OF HAD A POSITIVE SCORE BUT AGAIN THE ABSOLUTE VALUE OF THAT IS ALWAYS LESS THAN THE CHROMOSOME ARM SCORES SHOWN HERE. SO WE THINK THAT ANEUPLOIDY IS ACTUALLY DRIVING THIS BUT -- DRIVING THE SIGNATURE, BUT IT IS JUST A CORRELATION. SO IN ORDER TO TRY TO TEST THAT, WHETHER IT ACTUALLY WAS DRIVING, WE WENT TO A MOUSE CANCER MODEL -- SORRY, FIRST WE DID A HUMAN CELL ANALYSIS. WE'LL GET TO THE MOUSE IN A MINUTE. WE TOOK THESE EPITHELIAL CANCER CELLS THAT WERE HACKING P53. AND -- LACKING P53. THESE ARE PRIMARILY DIPLOID CELLED. WE WANTED TO MAKE ISO GENIC ANEUPLOID CELLS OUT OF THESE. WE INHIBITED THE MITOTIC CHECK POINT USING CHECK POINT INHIBITOR, AND WHAT YOU CAN SEE IS THAT THE CHROMOSOME NUMBER NOW GOES FROM 46 TO A LOT OF DIFFERENT ANEUPLOID NUMBERS, BOTH LOSS AND GAIN. AND THEN WE ASKED WHETHER OR NOT T CELLS COULD PRODUCTIVELY INTERACT WITH THESE. SO EACH OF THESE, THE PARENT CELL WAS EXPRESSING A PRECURSOR PROTEIN FOR A PROTEIN THAT GENERATES A T CELL EPITOPE. WE HAD THE CD8T CELLS, THIS IS HIV PROTEIN THAT WAS SPECIFIC FOR THIS EPITOPE. AND YOU CAN SEE THAT IN THE ANEUPLOID CELLS THEY EXPRESSED THE SAME LEVELS. THEN WE LOOKED AFFIDAVIT AT CD8 MEDIATED CELL KILLING. IF WE ADDED A NON SPECIFIC T CELL, THEY LOOK ABOUT THE SAME. AND THIS IS WHETHER THEY'RE TREATED -- CONTROL OR REVERSEN siRNA MED 2 OR SCRAMBLE. WHAT YOU SEE IS THAT THE DIP PLOYED CELLS NOW, IF YOU ADD THE T CELL SPECIFIC, THIS IS KILLING, FRACTION OF KILLING. THEY'RE KILLED VERY EFFICIENTLY. THE ANEUPLOID CELLS NOW HAVE A DEFECT IN KILLING. SO WE THINK THAT NOT ONLY DOES ANEUPLOIDY CORRELATE WITH INFILTRATION BUT IT'S IMPAIR THE IMMUNE RESPONSE. WE ALSO WENT TO A MOUSE MODEL WHERE WE TOOK A TUMOR THAT EVOLVED IN A BLACK 6 BACKGROUND. WE MODEL IT -- IT WAS ESSENTIALLY DEPLOYED. WE MODEL IT ANEUPLOID. AND YOU CAN LOOK HOW THEY GROW IN CULTURE. THE DIPLOID CELLS GROW A LOT BETTER THAN THE ANAP. WE KNOW ANEUPLOID CELLS ARE SICKER. A LOT OFF WORK FROM [INDISCERNIBLE] LAB HAS SHOWN THIS. THEN WE TOOK THESE TUMOR LINES, THE DIPLOID AND ANAPVERSION. WE PIT THEM INTO WILDTYPE MICE AND WE SEE HOW THEY GROW IN THE RAG DEFICIENT MICE. SO THEY'RE MUCH -- THE TUMOR VOLUME GROWS MORE SLOWLY. WE PUT THEM, THEN, INTO A WILDTYPE MOUSE THAT HAD A WILDTYPE IMMUNE SYSTEM. AND NOW YOU CAN SEE THAT THE -- THOSE TWO TUMORS GROW ABOUT THE SAME RATE. SO THE WILDTYPE OR DIPLOID TUMORS TAKE A MUCH BIGGER HIT THAN THE ANEUPLOID TUMORS. WHEN YOU TAKE THESE TUMORS OUT AND LOOK AT THE IMMUNE INFILTRATION, LOOKING AT INFILTRATING T CELLS YOU CAN SEE THE DIPLOIDS HAVE MORE THAN THE ANAPCELLS. ANEUPLOID CELLS. THESE ALL CD8T CELLS. THIS IS RECAPITULATING WHAT WE THOUGHT WAS HAPPENING IN VITRO, IS NOW HAPPENING IN VIVO. SO WE THINK THESE THINGS ARE REALLY CAUSAL. WHAT'S THE SIGNIFICANCE OF THIS, ABILITY TO ESCAPE THE IMMUNE SYSTEM. WE ASKED WHETHER OR NOT ANAPY CAN IMPACT THERAPIES SUCH AS IMMUNE CHECK POINT BLOCKADE. SO WE LOOKED AT [INDISCERNIBLE], TREATED WITH CTLA4 IN TWO DIFFERENT CLINICAL TRIALS WHERE THEY LOOKED AT THE GENOME, OF THE TUMORS FOR THESE PATIENTS. WHAT YOU CAN SEE, IF YOU JUST DIVIDE THESE TUMORS NOW INTO THE TOP AND LOWER 50% OF ANEUPLOIDY, AND THEN PLOT THEIR SURVIVAL IN RESPONSE TO ANTICTLA4 TREATMENT, WHAT YOU CAN SEE IS THAT THE HIGH ANEUPLOIDY TUMORS PATIENTS DON'T RESPOND NEAR AS WELL AS THE LOW ANEUPLOID TUMORS. THIS IS FROM THE VAN ALLEN STUDY, LEVI'S LAB. SMALLER STUDY, SCHNEIDER ET AL. WE GET A VERY SIMILAR RESPONSE. IF WE LOOK NOW WHAT THE MUTATIONAL BURDEN DOES, IT'S JUST THE OPPOSITE. IF YOU HAVE HIGH MUTATION LOAD, YOU'RE SLIGHTLY BETTER AT RESPONDING. IT DOES REACH STATISTICAL SIGNIFICANCE, UNLIKE ANEUPLOIDY. IF YOU ADD THESE TOGETHER, YOU GET AN EVEN BETTER SCORE. SO IT LOOKS LIKE THEY'RE LARGELY ACTING INDEPENDENTLY OF EACH OTHER. AND THAT'S TRIIN BOTH OF THESE -- TRUE IN BOTH OF THESE SETS. LET ME JUST SUMMARIZE WHAT I TOLD YOU TODAY. FIRST OF ALL, THAT WE THINK THAT THE BALANCE BETWEEN THESE GROWTH PROMOTING GO GENES AND THE GROWTH INHIBITORY STOP GENES, TUMOR SUPPRESSER LIKE, ALONG THE CHROMOSOMES ULTIMATELY DETERMINES THE SELECTIVE PRESSURE THAT DRIVES SOMATIC COPY NUMBER VARIATIONS THAT YOU SEE IN CANCERS. THIS IS ALL DUE TO CUMULATIVE GENE DOSAGE EFFECT OF MULTIPLE DIFFERENT GENES THAT HAVE SMALL EFFECTS BUT COLLECTIVELY THEY ACTUALLY HAVE A ROLE. THE OTHER POINT I WANTED TO MAKE IS THAT IN DIFFERENT CELL TYPES THERE IS AN UNDER LYING EPIGENETIC OR REGULATORY ARCHITECT THAT CONTROLLED PROLIFERATION RESPONSIVENESS. ULTIMATELY, THE PATTERNS AND SOMATIC COPY NUMBER ALTERATIONS IN TUMORS. I GAVE YOU -- PRESENTED EVIDENCE THAT ANEUPLOIDY DRIVES 2 HALLMARKS OF CANCER, PRO LEGISLATIVERATION AND IMMUNE EVASION. HIGH ANEUPLOIDY PROMOTES CELL PROLIFERATION BUT INHIBITS IMMUNE INFILTRATION. THE OPPOSITE IS TRIOF LOW ANEUPLOIDY, AND THAT -- TRUE OF LOW ANEUPLOIDY. WHAT'S INTERESTING, THE PROLIFERATION SIGNATURE IS PREDICTED BY FOCAL EVENTS GREATER THAN ARM EVENTS, BUT THE IMMUNE EVASION IS PREDICTED LARGELY BY WHOLE CHROMOSOME AND WHOLE CHROMOSOME ARM EVENTS. WE THINK THAT'S A DISTINCTION MECHANISM. WE CAN TALK IN THE QUESTION AND ANSWERS ABOUT WHAT THAT IS. AND FINALLY, THAT ANAPY IS A -- ANEUPLOIDY IS A STRONGER PREDICTER THAN MUTATION LOAD FOR IMMUNE INFILTRATION AND BETTER PREDICTS THE OUTCOME OF IMMUNOTHERAPY. THIS IS IN TRIALS JUST FOR CTLA4, REMAINS TO BE SEEN IF THIS IS ALSO GOING TO BE THE CASE FOR IMMUNOTHERAPY WITH ANTI-PD1 ANTIBODIES. AND WE'RE EAGER TO DO THOSE ANALYSIS. SO LET ME JUST STOP AND THANK THE PEOPLE WHO DID THIS WORK. THE INITIAL WORK ON THE [LIST OF NAMES]. ALSO THE WORK ON IF CORRELATION WITH THE ABILITY OF ANEUPLOIDY TO DRIVE CELL PROLIFERATION SIGNATURES AND IMMUNE EVASION SIGNATURES WAS SPEAR HEADED BY [LIST OF NAMES] THANK YOU FOR YOUR ATTENTION. I'M HAPPY TO TAKE ANY QUESTIONS. [APPLAUSE] >> HI, STEVE, VERY NICE TALK. >> THANK YOU. >> SO YOU WHO YOU THINK THE IMMUNE SYSTEM RECOGNIZES THERE IS CHROMOSOME IMBALANCE? >> I THINK THAT THE IMPLICATION IS THAT THE T CELLS ARE NOT RECOGNIZING THE TUMOR AS WELL. THEY DON'T KILL STEM CELLS STEM CELL, AT LEAST IN THE MODEL SYSTEM WE DEVELOPED. THE IDEA IS IN ORDER TO GET TUMOR CELL TUMORS INFILTRATED BY THE IMMUNE SYSTEM IT'S A FORWARD FEEDING LOOP. IF THE T CELLS GET IN AND KILL CELLS THOSE CELLS GET PRESENTED TO PRESENTLING CELLS, STIMULATING MORE T CELLS TO PROLIFERATION AND BECOME ACTIVE. THEY KILL MORE CELLS. IT'S A POSITIVE FEEDING LOOP. IF YOU CAN DAMPEN THAT AT ANY STAGE, YOU'LL REDUCE THE IMMUNE INFILTRATION. WE THINK THERE WILL BE AN ISSUE WITH ANTIGEN PRESENTATION, ONE IDEA IS THAT THE -- AND BY NO MEANS THE ONLY POSSIBLE EXPLANATION, IS THAT NOW YOU'RE MAKING THE WRONG NUMBERS OF A LOT OF WILDTYPE PROTEINS. THEY'RE SORT OF FLOODING THE PROTEOSOME AND BEING PRESENTED. THEREFORE, THE PERCENTAGE OVER NEO EPITOPES THAT GET PRESENTED IS REDUCED. BECAUSE THEY'RE BEING OUT-COMPETED TO SOME DEGREE. AND I THINK EVEN A 50% REDUCTION IS LIKELY TO HAVE A SIGNIFICANT EFFECT, AND THE REASON FOR THAT IS THAT THERE IS A WAY TO REDUCE PRESENTATION BY 50% BY MUTATING BETA 2 MICRO GLOBULIN, THE PARTNER FOR MHC. FOR ALL MHCISTS OF CLASS ONE. IF YOU GIVE RID OF HALF OF THAT THAT'S SELECTED FOR A MUTANT IN CANCER ALL THE TIME. >> YOU HAD A DIFFERENCE IN GROWTH BETWEEN -- WHEN -- >> I'M SORRY? >> WHEN PRIMAR CELLS WERE ANEUPLOID, THEY GREW POORLY. THE TUMOR CELLS GREW MUCH BETTER IF THEY WERE ANEUPLOID >> THEY DIDN'T GROW BETTER. THEY GREW WORSE. >> THE TUMOR CELLS, THERE WAS A GROWTH. >> RELATIVE TO DIPLOID. >> WHEREAS PRIMARY CELLS, THERE WAS HA GROWTH DISADVANTAGE. >> RIGHT. >> HOW -- >> SO WHY DID THAT WORK? BECAUSE I THINK THERE IS TWO VARIABLES THAT ARE VARYING. ONE IS THEY'RE INTRINSIC ACT TO DIP PLIICATE. AND THE ANEUPLOID CELLS ARE REALLY BAD. SO THE DIPLOID CELLS ARE OUT-GROWING THEM. BUT NOW YOU LAY ON TOP OF THAT, THAT THE DIPLOID CELLS ARE BEING KILLED A LOT BY THE WILDTYPE IMMUNE SYSTEM. THAT REDUCES THEM TO THE SAME LEVEL. SO THEY'RE PROBABLY STILL PROLIFERATIONING GETTING BUT GETTING KILLED BY THE IMMUNE SYSTEM AND THEY CAN CONVERGE. SO I THINK THAT'S PROBABLY EXPLAINING IT, THE WAY WE THINK ABOUT THAT. >> I'M ASSUMING THAT MOST OF THE STUDIES YOU SHOWED WERE DONE ON HUMAN CELLS AND HUMAN CANCER LINES, IS THAT SO? >> YES. THAT'S -- ALL THE STATISTICAL UNLESS, EXCEPT THAT ONE MOUSE STUDY. >> THE ONE MOUSE STUDY, THEY'RE TOLOCALEROUS EXPRESSERS. THEY DON'T NECESSARILY REFLECT WHAT'S IN HUMAN. MY QUESTION IS THERE IS TWO MODELS, ONES A NAKED MOLE RAT, THE OTHER A BLIND MOLE RAT. ARE MAMMALS THE EXCEPTION TO THE RULE IF THEY DO NOT DEVELOP CANCER, AND THEY BOTH LIVE FOR A LONG TIME, ABOUT 30 YEARS OR SO IN COMPARATIVE. AND THEY HAVE UNIQUE METHODS OF TUMOR SUPPRESSION. HAVE YOU EVER THOUGHT OF PUTTING WITHIN YOUR ANALYSIS HOW THEY'RE DOING IT AND DO IT ON A WEIGHTED RELATIVE ANALYSIS WITH WHAT YOU HAVE? >> I DON'T THINK WE HAVE THE NECESSARY DATA ON THAT TO DO THE ANALYSIS. YOU COULD POTENTIALLY DO CELL LINE STUDIES. THEY DID RECENTLY FIND TWO CANCERS IN NAKED MOLE RATS. THEY FOUND TWO DIFFERENT CANCERS IN THE NAKED MOLE RAT RECENTLY. THEY DO GET SOME CANCERS. I MEAN THAT'S INTERESTING, WHETHER OR NOT THEY -- THE REASON THEY HAVE FEWER CANCERS HAS TO DO WITH, YOU KNOW, A BETTER IMMUNE SYSTEM. I THINK THAT'S AN OPEN QUESTION. BUT WE'RE NOT IN A POSITION RIGHT NOW TO TEST THAT. BUT IT'S AN INTERESTING QUESTION. >> OKAY. THANK YOU. >> SURE. >> HI. WERE ALL THE CELL LINES FROM WOMEN? >> WERE ALL OF THEM? >> YES. >> ALL THE BREAST LINES WERE. >> HOW ABOUT THE PANCREATIC ONES? >> NO. I DON'T KNOW WHERE -- IF THEY WERE FEMALE OR NOT. ACTUALLY, I SHOULD CHECK THAT. >> WOULD YOU EXPECT ANY DIFFERENCE IN THE GO GENES VERSES THE STOP GENES? >> I WOULDN'T EXPECT IT. BUT IF YOU DON'T DO THE EXPERIMENT YOU DON'T KNOW. >> I WAS JUST WONDERING, AND YOU MIGHT HAVE MENTIONED THIS DURING YOUR TALK, DID YOU AT ALL LOOK AT [INAUDIBLE] ANEUPLOIDY ON THE RATE AND LIKELIHOOD OF METABOLISM IN THESE TOMB -- METASTASES IN THESE TUMORS? >> NO. WE DIDN'T LOOK AT THAT. AND THE REASON IS THE MOUSE STUDIES ARE JUST REALLY RECENT, BUT ALL THE DATA THAT WE ANALYZED WAS ALL FROM PRIMARY TUMORS. SO THE DATA FOR THE METS ISN'T AVAILABLE, ALTHOUGH YOU COULD SAY INDIRECTLY SURVIVAL, YOU KNOW, OF -- IN THE IMMUNE THERAPY TRIALS IS LINKED TO THAT. BUT IT WASN'T REALLY UTILIZED, NO. >> GREAT TALK. I WANT TO GO WACK TO THE DARK MATTER OF THE CANCER GENOME YOU WERE TALKING ABOUT WHERE THE GO GENES AND STOP GENES ARE, BASICALLY [INDISCERNIBLE] HAPPENED TO BE MUTATED. HAVE YOU LOOKED AT WHETHER THE GO AND STOP GENES EFFECT THE TRANSFORMATION PHENOTYPE OF CELLS, ALONE OR IN COMBINATION WITH ANOTHER DRIVER. >> THE ACT TO GROW SOFT -- >> THAT'S RIGHT, YEAH. >> NO, WE HAVEN'T DONE THAT SCREEN RECENTLY. WE DID IT FIVE OR TEN YEARS AGO WITH EARLIER VERSION OF THE LIBRARY BUT NO, WE HAVEN'T DONE THAT. WE'VE DONE SOME [INDISCERNIBLE] BUT NOT WITH THIS LIBRARY, BUT WITH SPECIFIC TUMOR SUPPRESSER ONCOGENE LIBRARIES AS, YOU KNOW, SOMETHING LIKE THAT. BUT NO. >> I STUDY GENETICS, CANCER HAVE KIDNEY -- CANCER OF THE KIDNEY. EARLIER I SAW 25 PATIENTS WITH DIFFERENT TYPES OF INHERITED FORMS OF KIDNEY CANCER. WE KNOW OF AT LEAST 18 GENES THAT CAUSE KIDNEY CANCER. WE HAVE ABOUT 40 IN ALL 3 PROJECTS THAT ARE SMGs. ABOUT 17 -- FOR DIFFERENT REASONS. WHEN WE LOOK AT THE TIMERS, AND WE WERE TALKING EARLIER TODAY AS WE WENT OVER OUR FILMS FOR OUR PATIENTS ABOUT YOUR TALK THIS AFTERNOON. I TOLD EVERY ONE IN OUR GROUP, YOU GOT TO COME TO HIS TALK. BUT ONE OF THE THINGS WE WERE SORT OF THINKING ABOUT WAS WE HAD SEEN -- WE CAN LOOK AT CGH AND DO ALL THAT. BUT WE'VE SEEN IN MANY OF OUR TUMORS, FOR EXAMPLE, AND I HAVE BEEN THINKING ABOUT THIS, YOUR WORK AND YOUR TALK TODAY KIND OF MAYBE CRYSTALLIZED A LITTLE BIT OF THIS MAYBE, IN THAT SO WE'RE TALKING ONE ORGAN, KIDNEY. AND WE HAVE THESE DIFFERENT GENES, VERY DIFFERENT KINDS. SOME ARE -- OUR FIRST GENE WAS VHL, LOSS OF FUNCTION GENE. OUR SECOND WAS MET, GAIN. AND WE SEE THAT. SO OUR SENSE A LITTLE BIT IS MAYBE WHAT WE'RE SEEING IS HAPLOOF THESE OTHER GENES. LIKE WE CAN -- THAT WE CONSIDER GENES THAT CAUSE KIDNEY CANCER. LIKE [INDISCERNIBLE], AND THAT WE THINK -- WE'RE WONDERING IF IT MAKES SENSE TO THINK THE COMBINATION OF THOSE GENES THAT DRIVE KIDNEY CANCER COULD IN AN INDIVIDUAL CANCER, FOR EXAMPLE, COULD GIVE US A MORE SORT OF AGGRESSIVE PHENOTYPE, OR MAYBE BE INVOLVED IN TRANSFORMATION TO BEGIN WITH. DO YOU THINK THAT MAKES SENSE? >> I DO THINK SO. PERSONALLY, I THINK THAT ALL THE SPORADIC TUMOR SUPPRESSERS ARE HAPLOINSUFFICIENT. YOU LOSE BOTH ALLELES IT'S MORE POTENT. CERTAINLY, THEN, IF YOU HAVE COMBINATIONS OF THEM, PRESENT IN A CERTAIN TIMER, THAT SHOULD BE EVEN, YOU KNOW, MORE AGGRESSIVE. SO I THINK IT MAKES SENSE. >> WHEN YOU HAVE, FOR EXAMPLE, HIGH COPY NUMBER AND IN ONE OF OUR TYPES OF TUMORS WE HAVE A VERY HIGH COPY NUMBER OF CHROMOSOME 7. WE KNOW THAT THE HEREDITY VERSION OF THAT CANCER IS MUTATION. GENE 7, WHICH IS MET, A GAPE OF FUNCTION GENE OBVIOUSLY. HOW DO YOU GO ABOUT FIGURING WHEN YOU HAVE A WHOLE GAIN OF WHOLE CHROMOSOME. WE SEE IN THE SPORADIC TUMORS 82% HAVE MULTIPLE COPIES OF CHROMOSOME 7. HOW DO YOU FIGURE OUT -- WE CALL THAT DARK MATTER. GLIT. >> HOW DO YOU GO FROM WHERE WE ARE NOW TO FIGURE OUT WHAT GENE IT IS. >> THIS IS A PROBLEM A LOT OF PEOPLE HAVE BEEN WORKING ON. SCOTT LOWE HA DONE A LOT OF WORK ON IN THIS LIVER CANCER. THE STRATEGY HE TOOK WAS TO TAKE THE GENES AND OVER-PRODUCE THEM TO SEE IF THEY GIVE YOU, INDIVIDUALLY, TO SEE IF THEY GIVE YOU A CANCER CONSISTENT PHENOTYPE, OR TO, THEN, KNOCK DOWN GENES ALONG THE CHROMOSOME TO SEE IF THEY REDUCE THE CANCER PHENOTYPE IN THE MOUSE MODEL. SO I THINK THAT'S THE ONLY WAY YOU CAN REALLY PROVING THAT ANY INDIVIDUAL GENE IS GOING TO BE DRIVING. I'D ALSO LOOK AT THE FOCAL EVENTS ON THAT BECAUSE IF YOU DO FIND THAT SOME CANCERS DON'T OVER-PRODUCE, DON'T GAIN THE TOTAL CHROMOSOME, BUT THAT THE FOCAL EVENTS ARE THERE, THOSE FOCAL EVENTS ARE IMPORTANT IN DRIVING THAT WHOLE EVENT OUTSIDE OF MET. >> OKAY. >> ALL RIGHT. SING I'D LIKE TO THANK STEVE FOR THE VERY SKIMULATING LECTURE. >> -- STIMULATING LECTURE. THERE IS A RECEPTION IN THE LIBRARY FOR THE -- THOSE THAT WANT TO ATTEND. STEVE WILL STAY A LITTLE BIT BEFORE HE CATCHES HIS PLANE. YOU CAN TALK TO HIM THERE.