>> GOOD AFTERNOON, AND YOU SHOULD HAVE ALL GOTTEN AN E-MAIL FROM ME LAST WEEK, IF ANY OF YOU WERE INTERESTED IN ATTENDING THE TRACO COURSE, YOU CAN SEND ME AN E-MAIL AND TELL ME WHICH ONES YOU HAVE. TODAY WE HAVE WYNDHAM WILSON. HE CAME TO THE NCIA IN 1984 AND SET UP THE METABOLISM BRANCH, HEAD OF THE LYMPHOMA THERAPEUTIC SECTION. WYNDHAM. >> OKAY. >> ALL RIGHT, SO WHAT I'M GOING TO DO IS I'M GOING TO FIRST START OUT BY JUST GIVING YOU KIND OF A GENERAL REVIEW OF WHAT LYMPHOMAS ARE AND THEN I'M GOING TO SPEND MOST OF THE TALK TALKING ABOUT SOME OF THE SCIENCE THAT I'VE BEEN WORKING ON AND TRYING TO USE THAT TO TRANSLATE INTO NEWER THERAPISTS. I THINK THAT'S GOING TO BE OF MORE INTEREST TO YOU. BUT FOR THOSE OF YOU WHO JUST WANT A VERY BRIEF PRIMER, LYMPHOMAS ARE AROUND THE FOURTH MOST COMMON TYPE OF TUMORS OUT THERE. A LITTLE MORE COMMON IN MEN THAN IN WOMEN, BUT VERY VERY CLOSE. THIS IS A DISEASE THAT OCCURS AROUND 70,000 CASES EACH YEAR AND IT IS A DISEASE WHERE KIND OF THE MEDIAN AGE FOR MOST OF THEM ARE IN THE 60'S AND SO WHEN YOU'RE OVER 60, THAT'S WHEN THE RATES REALLY GO UP, AROUND ABOUT EIGHT TIMES HIGHER INCIDENCE IN FOLKS OVER 65 VERSUS UNDER 65. BUT LYMPHOMAS DO OCCUR AT ALL AGE GROUPS. THEY OCCUR IN FIVE YEAR OLDS AND THEY CAN OCCUR IN 90 YEAR OLDS. NOW ONE THING THAT'S NOT BEEN WELL UNDERSTOOD AND IT'S STILL NOT UNDERSTOOD IS WHY THE INCIDENCE OF LYMPHOMAS HAS BEEN RISING OVER THE LAST 20 YEARS. NOW THIS HAS BEEN MORE OR LESS LEVELING OUT, BUT FOR AROUND THE 20 YEAR PERIOD, THE DATABASE CLEARLY SHOWS THAT THE INCIDENCE WAS GOING UP. AND IT ACTUALLY WAS RISING AT A PRETTY ROBUST RATE AT AROUND 3-5% PER YEAR. MOST OF US THINK IT'S RELATED TO ENVIRONMENTAL EXPOSURES BUT NONE OF THIS HAS REALLY BEEN PROVEN. WE DO NO IF ASSOCIATED WITH HIV, HOWEVER, THE NUMBER OF CASES OF HIV LYMPHOMA IN THIS COUNTRY DON'T EVEN COME CLOSE TO REALLY COUNTING FOR THIS RISE. NOW, THERE ARE A WHOLE HOST OF DIFFERENT RISK FACTORS. OF THESE ARE OF EXTERNAL, BUT WE ALSO KNOW THAT THERE ARE CERTAIN GENETIC RISK FACTORS AS WELL IN TERMS OF VARIOUS SNIPS AND WE ALSO KNOW THERE ARE A VARIETY OF INHERITED CONDITIONS THAT ARE ASSOCIATED WITH LYMPHOMA AS WELL. AND OF COURSE THE ONES THAT LEAD THE LIST WOULD BE THOSE THAT ARE ASSOCIATED WITH IMMUNODEFICIENT Z PATIENTS WITH DISEASES OR A VARIETY OF OTHER DISORDERS WHERE IMMUNE DEFICIENCY IS AT THE ROOT, HAVING INCREASED INCIDENCE. ALSO INTERESTINGLY IS THAT IMMUNE STIMULATION, AUTOIMMUNE ESTIMATION IS ALSO ASSOCIATED WITH IT. YOU HAVE TWO THINGS GOING ON HERE, THE IMMUNO SURVEILLANCE WHICH IS PART OF CLEARING ABNORMAL CELLS OUT AND WHEN THAT GOES BAD YOU SEE INCREASED LYMPHOMAS. AND SOME OF THE AUTO, SOME OF THE IMMUNODEFICIENCY LYMPHOMAS IS ALSO ASSOCIATED WITH SUSCEPTIBILITY TO INFECTIOUS AGENTS SO THAT'S A MIXED PICTURE. ON THE OTHER HAND AUTO IMMUNE STIMULATION IS ASSOCIATED WITH STIMULATING YOUR B CELLS WHEN THEY GET STIMULATED, THEY ARE TURNING OVER AND WE BELIEVE ARE AT GREATER RISK OF GETTING GENETIC EFFECTS. NOW THERE CAN ALSO BE EXTERNAL CAUSES OF IMMUNE STIMULATION AS WELL. HIV IS A WELL-KNOWN ONE. ANOTHER ONE WOULD BE SPREW, WHICH IS A GENETIC AUTOIMMUNITY AGAINST CERTAIN ANTIGENS. ALSO, THERE ARE CERTAIN INFECTIOUS GI ETIOLOGIES THAT CAN BE ASSOCIATED WITH INCREASED STIMULATION AS WELL. NOW, CHEMICAL EXPOSURE'S BEEN KNOWN FOR A VERY LONG TIME. I THINK MOST OF YOU REALLY HAVE HEARD THAT IT'S ASSOCIATED WITH PESTICIDES, AGENT ORANGE, OF COURSE, BECAUSE IT WAS ASSOCIATED WITH THE VIETNAM WAR WAS THE ONE WE THINK ABOUT MOST. BUT IT ACTUALLY TURNS OUT THAT EXPOSURE TO ORGANIC SOLVENTS IS ACTUALLY A HIGHER RISK. SO CHEMICALS CAUSE IT AND THEN OF COURSE THERE'S A WHOLE HOLES OF INFECTIOUS ETIOLOGIES. SOME IT ARE DUE TO IMMUNODEFICIENCY. OTHER ONES CAN LEAD TO THIS AUTOIMMUNE STIMULATION AND THEN YOU CAN HAVE AN INTERPLAY OF THE TWO. SO WHEN YOU LOOK AT A NUMBER OF THE DIFFERENT INFECTIOUS ETIOLOGIES ASSOCIATED WITH VARIOUS DIFFERENT SUBTYPES OF LYMPHOMA, WE KNOW THAT EBB IS ASSOCIATED WITH BURKITT'S LYMPHOMA, HUGE KIN'S DISEASE, POST TRANSPLANT LPD OR LYMPHOPROLIFERATIVE DISEASE IS ACTUALLY MOSTLY EBV PROCESS WHERE YOU HAVE ACTIVATION OF EBV. HIV IS NOT DIRECT, IT'S INDIRECT IN TERMS OF WHAT IT DOES TO BOTH IMMUNITY AS WELL AS STIMULATING OF B CELLS. THE FIRST VIRUS EVER KNOWN TO BE ASSOCIATED WITH HUMAN DISEASE WAS IN FACT HTLD1 WHICH MANY OF YOU MAY KNOW -- FOUND AND THAT'S THE CAUSE OF SOMETHING CALLED THE CELLS WITH LEUKEMIA LYMPHOMA. OTHER VIRUSES. HHV8 -- A MAJOR CAUSE OF GI ULCERS IS ALSO YES ASSOCIATED WITH MUCOSAL LYMPHOMAS AND PATHOGENS THAT ARE ASSOCIATED WITH SOME MEDITERRANEAN GI DISORDERS. SO, HOW DO WE THINK ABOUT LYMPHOMAS. IT'S NOT A SINGLE TUMOR TYPE. MAY HAVE READ I THINK IT WAS IN THE NEW ENGLAND JOURNAL WHERE THEY RECENTLY THROUGH THE GENOME PROJECT HERE AND A VARIETY OF OTHER SOURCES IDENTIFIED BREAST CANCER AS BEING FOUR DIFFERENT TYPES. LYMPHOMAS ARE 60 TYPES AND COUNTING. NOW WE'VE KNOWN THAT LYMPHOMAS ARE A LOT OF DIFFERENT TYPES BUT AS WE'RE BEING ABLE TO EVEN DRILL DOWN MOLECULARLY WHAT USED TO BE FELT TO BE A SINGLE TYPE ARE REALIZING THOSE ARE MULTIPLE TYPES AS WELL. AT THE HEART OF ALL THIS IN TERMS OF TRYING TO UNDERSTAND HOW YOU WANT TO DEVELOP THERAPY, YOU HAVE TO HAVE A GOOD PATHOLOGIC CLASSIFICATION SYSTEM THAT'S GOING TO BE ABLE TO ALLOW YOU TO REPRODUCIBLY IDENTIFY THOSE TYPES. AND SO THIS HAS BEEN A MAJOR AREA THAT PEOPLE HAVE WORKED ON. THERE HAVE BEEN MANY DIFFERENT CLASSIFICATION SYSTEMS OVER THE YEARS. JUST IN TERMS OF A VERY SIMPLE PRIMER. HOW DO WE CATEGORIZE LYMPHOMAS. THERE WAS A TIME WE COULDN'T DIFFERENTIATE BETWEEN T CELL AND B CELL LYMPHOMAS. THEY WERE JUST CALLED LYMPHOMAS. NOW WE CAN IMMUNOTYPE THEM SO WE IDENTIFY THEM AS T CELL TYPE OR B CELL TYPE. THREE QUARTERS OF ALL LYMPHOMAS, 80% ARE DERIVED FROM A B LYMPHOCYTE WHERE THE REMINDER ARE FROM T CELLS ARE SO CALLED NATURAL KILLER CELLS. WE ALSO WILL HAVE A NAME BY A LYMPH NODE THEY ARE ACTUALLY FOUND. THE NAME IS AFTER ALL SO BASED ON WHAT THE CELLS LOOK LIKE SO THE CYTOLOGIC APPEARANCE OF THE CELL. AND MANY OF YOU HAVE HEARD THAT WE THINK OF LYMPHOMA BEING HODGKINS VERSUS NON-HODGKINS LYMPHOMAS. WE STILL ACTUALLY USE THOSE TWO GENERAL CATEGORIES ALTHOUGH I THINK IT'S REALLY JUST A MATTER OF TIME BEFORE THESE ARE ACTUALLY DROPPED. NOW WHY DO WE ACTUALLY DO THAT? THE REASON WE DO IT WAS BECAUSE HODGKINS DISEASE WHICH WE NOW CALL HODGKINS LYMPHOMA, UP UNTIL AROUND 12 YEARS AGO, WE DIDN'T EVEN KNOW THAT IT WAS DERIVED FROM A LYMPHOCYTE. AND THE REASON FOR THAT IS ONE OF THE PRIMARY CHARACTERISTICS OF HODGKINS DISEASE IS THAT IT HAS DOWN REGULATED THE B CELL PROGRAM. AND SO YOU DON'T SEE THE TYPICAL B CELL ANTIGENS ON THE SURFACE, WHEN YOU GET IMMUNOPHENOTYPING THEY DIDN'T SEE THE NORMAL B CELL AMOUNT JUDGES SO THEY COULDN'T IDENTIFY IT AS NECESSARILY BEING A B CELL. YOU SAY WHAT ABOUT DOING IMMUNOGLOBULIN ARRANGEMENTS. THEY TRIED THOSE BUT ONE OF THE THINGS CHARACTERISTIC OF HODGKINS IS THERE ARE A FEW CELLS SCATTERED AMONG A C, T CELLS, B CELLS, NEUTROPHILS SO WHEN YOU GO AHEAD AND DO PCR YOU OFTEN WOULD NOT BE ABLE TO PICK UP A MONOCLONAL SLIDE DUE TO A DELITTLAL EFFECT. WE HAVE IDENTIFIED LYMPHOMA ACCORDING TO MOLECULAR FEATURES INCLUDING SPECIFIC GENES AND THEN THERE'S ALWAYS SOME OVERLAY WITH HOW THE, WHAT THE CLINICAL PRESENTATION IS AND WHAT THE DISEASE SITES ARE. SO IF WE THINK ABOUT THE FREQUENCY NON-HODGKINS LYMPHOMAS ARE A LITTLE OVER 80% OF ALL LYMPHOMAS WHEREAS HODGKINS YOU CAN SEE IS AROUND 15-20%. EVEN HODGKINS LYMPHOMA IS NOT A SINGLE DISEASE TYPE, IT HAS BEEN SUBDIVIDED INTO ABOUT FIVE DIFFERENT TYPES BUT WE'RE NOT REALLY GOING TO -- -- BUT THERE ARE ACTUALLY MANY DIFFERENT TYPES. DUE TO THEIR RARITY, THE WHOLE GROUP OF THEM REALLY ONLY COMPRISING AROUND 7%, IT'S REALLY BEEN A VERY SLOW PROCESS TO REALLY TRY TO IDENTIFY THE VARIOUS DIFFERENT TYPES OF PERIPHERAL T CELL LYMPHOMAS. OTHER TYPES, MANY OF YOU HAVE PROBABLY HEARD OF COL, IT'S COUNTERPART IS SOMETHING CALLED SMALL SPECIFIC LYMPHOMA, THE SAME DISEASE BUT IT PRESENT AS A LYMPHOMA'S MASS, PRIMARILY AS OPPOSED TO A LEUKEMIC PHASE. OTHER TYPES OF LARGE CELL LYMPHOMAS ARE MEDIAL TYPES OTHERS. THIS IS WHAT A NORMAL LYMPH NODE LOOKS LIKE. IT HAS A SPECIFIC ARCHITECTURE, IT IS MADE UP OF A SERIES OF GERMINAL CENTERS WHERE THE GERMINAL CENTER REACTION TAKES PLACE. AROUND THE GERMINAL CENTER, THERE ARE THESE MANTEL ZONES AND MARGINAL ZONES AND LYMPHOMAS ARE CALLED -- HAVE GENERALLY TAKEN THEIR NAME FROM CELLS FROM P LYMPHOCYTES THAT ARE SPECIALIZED AND ARE ACTUALLY FOUND WITHIN THESE VARIOUS ZONES. OTHER LYMPHOMAS LIKE LARGE CELL LYMPHOMA TAKE ITS NAME FROM THE FACT THE CELL LOOKS LARGE. IT'S A HODGEPODGE OF DIFFERENT NAMES WITH DIFFERENT ORIGINS. SO ONE OF THE MOST IMPORTANT THING IS REALLY TO UNDERSTAND SOME OF THE BIOLOGY THAT UNDERLIES A NORMAL P CELL. YOU ALL KNOW THAT B CELLS GO THROUGH A SERIES OF B CELLS DIFFERENTIATION, PRO B, PRE B. DISEASES ARE ACUTE LYMPHOBLASTIC LEUKEMIA, A DISEASE THAT CHILDREN GET OR LYMPHOBLASTIC LYMPHOMA, A DISEASE THAT ADULTS CAN GET BUT ALSO CHILDREN MOSTLY. SO THOSE ARE NOT THE DISEASES WE'RE GOING TO BE DISCUSSING TODAY, WE'RE GOING TO BE DISCUSSING DISEASES THAT ARE REALLY MUCH FURTHER ALONG IN THEIR DIFFERENTIATION PATHWAY. MOST LYMPHOMAS ACTUALLY DERIVE FROM A B CELL THAT IS STILL WITHIN THE GERMINAL CENTER. NOW THIS IS A KEY AREA BECAUSE THERE IS A LOT OF PROLIFERATION THAT IS GOING ON WITHIN THESE B CELLS. THEY ARE STAGED THERE TO BE PROLIFERATING AND TURNING OVER AND WAITING FOR AN ANTIGEN TO IMMORTALIZE THEM OR GO THROUGH A SPAIN TAIN JUST APOPTOSIS. IF WE LOOK AT THE VARIOUS LYMPHOMAS DERIVED WE NOW FROM A GERMINAL B CELL, THE SECOND MOST COMMON TYPES OF LYMPHOMA BIRKETT LYMPHOMA IS DERIVED FROM A LYMPHOMA CENTER B CELL. ONCE A NORMAL B CELL LEAVES, THEY GO ON TO BEGIN TO DIFFERENTIATE TOWARD A PLASMA BLASTIC B CELL AND ULTIMATELY A PLASMA CELL, AND IT IS THE SO-CALLED CLOSED TERMINAL CENTER OR ACTIVATED B CELLS FROM WHICH ANOTHER LARGE TYPE CELL WHICH WE WILL DISCUSS. CLL HAS BEEN AN UNCLEAR DISEASE, SOME OF THEM PROBABLY ARE DERIVED FROM A B CELL THAT HASN'T GONE THROUGH THE GERMINAL CENTER REACTION WHERE YOU HAVE A LOT OF SEMANTIC HYPER MUTATION GOING ON, WHERE AS OTHERS HAVE GONE THROUGH A GERMINAL CENTER REACTION BECAUSE YOU CAN FIND EVIDENCE OF SCHEMATIC HYPER MUTATIONAL WITHIN THE IMMUNOGLOBULIN GENES. T CELLS SIMILAR DIAGRAM CAN BE MADE FOR THEM AS WELL. MOST OF THE T CELL LYMPHOMAS ARE ACTUALLY THOSE THAT HAVE ACTUALLY GONE THROUGH THALAMIC REACTION. T CELLS THAT DO REACT TO AN ANTIGEN, AND MOST T CELL LYMPHOMAS ARE DERIVED FROM THE SO-CALLED PERIPHERAL T CELLS WHEREAS THERE ARE LYMPHOBLASTIC LYMPHOMA T CELL TYPE THAT IS A LITTLE BIT EARLIER OR THERE IS ALSO A T CELL AOL. MOST OF THESE EARLIER DISEASES LYMPHOBLASTIC OR MONO BLASTIC AOL ARE ACTUALLY DERIVED FROM A PRECURSOR B CELL. NOW, HOW DO WE FUNCTIONALLY IDENTIFY WHAT TYPES OF TUMORS WE'RE DEALING WITH? WELL WE FUNCTIONALLY DO IT BY GOING DOWN AND ASKING DR. ELAINE JAFFEY TO LOOK AT IT. THEY DO A WHOLE SERIES OF IMMUNOPHENOTYPING OF THE TUMOR AND THERE ARE DIFFERENT IMMUNOPHENOTYPES FOR DIFFERENT TYPES BUT THERE'S A LOT OF OVERLAP SO IT'S NOT JUST A MATTER OF LOOKING TO SEE WHAT THE IMMUNOPHENOTYPE IS, YOU HAVE TO PUT THAT IN CONCERT WITH THE CLINICAL PRESENTATION WITH WHAT THE TUMOR CELL LOOKS LIKE AND WITH CERTAIN MOLECULAR NORMALITIES. THERE ARE CERTAIN MOLECULAR PRENLDZ ABSOLUTELY SEE SEVEN FOR THE DIAGNOSIS. THE TWO, THE THREE THAT ARE ABSOLUTELY ESSENTIAL WOULD BE FOR BIRKETT YOU HAVE TO HAVE A MC TRANS . BUT IF YOU HAVE A MIC DOES THAT MAKE IT BIRKETT, NO. THEY CAN HAVE MIC BUT SO ARE -- ALCL. YOU DON'T HAVE TO BUT WE ARE NOW BEGINNING TO REALIZE THAT PROBABLY TO HAVE TRUE AN OWE PLASTIC LARGE CELL LYMPHOMA YOU NEED THE AL KINASE TRANSLOCATION. FINALLY IN ORDER TO HAVE MAMMAL CELL LYMPHOMA, YOU HAVE TO HAVE THE MCL ONE TRANSLOCATION 1114. THOSE ARE THE THREE LYMPHOMAS THAT HAVE TO IN MY VIEW HAVE THOSE TRANSLOCATION IN ORDER TO BE CALLED THOSE TUMOR TYPES. SOME PEOPLE WOULD ARGUE HERE BUT I THINK THAT'S JUST A MATTER OF MISDIAGNOSIS. SO IF YOU HAVE SOMEBODY COMING INTO YOUR CLINIC AND YOU HAVE A LUMP AND THEY TAKE IT OUT AND YOU FIND LYMPHOMA, WHAT DO YOU DO? THIS IS ALL VERY BORING FOR YOU BUT LET'S JUST CAPE THIS VERY -- KEEP THIS VERY QUICK. YOU DO A PHYSICAL, BLOOD WORK, A CT SCAN, YOU LOOK AT THE MARROW AND YOU ESSENTIALLY STAGE. YOU ASK YOURSELF BASED ON THESE TESTS WHERE HAS THESE DISEASE GONE TO. LYMPHOMA BY DEFINITION IS DERIVED FROM A B OR A T LYMPHOCYTE, WHAT THESE DO FOR A LIVING IS THEY CIRCULATE. SO THIS IS NOT LIKE SOLID TUMORS WHICH COME FROM AN ISOLATED, FROM AN ISOLATED ORGAN WHERE YOU CAN CURE THEM BY TAKING IT OUT EARLY BEFORE IT ACTUALLY SPREADS. IN LYMPHOMA, YOU ALMOST NEVER HAVE ANY BENEFIT FROM DOING SURGERY. NOW, LIKE EVERYTHING, NEVER SAY NEVER BUT THE TIMES THAT IT'S USEFUL IS SO INFREQUENT THAT YOU CAN ESSENTIALLY ASSUME THAT IT NEVER HELPS TO TAKE SOMETHING OUT. SO THESE ARE DISEASES THAT ARE FELT TO BE DISSEMINATED EARLY ON. HOWEVER, THERE ARE STAGE ONE MEANING THAT IT'S LOCALIZED TO A SINGLE AREA WHERE IT'S HARD TO FIND THAT IT'S DISSEMINATED. BUT IF YOU TRY A LOCAL THERAPY AND THEY HAVE A TYPE OF LYMPHOMA THAT CAN BE CURED, THE LOCAL THERAPY ALMOST NEVER WORKS IN A CURATIVE FASHION. SO YOU HAVE TO BE VERY CAUTIOUS. NOW, HOW DO WE STAGE. STAGING IS REALLY DONE BY HOW WIDELY SPREAD THE LYMPHOMA IS. NOW, I'VE JUST TOLD YOU THAT IF IT'S EARLY STAGE, IT'S PROBABLY ALMOST CERTAINLY OTHER PLACES BECAUSE WHAT THESE THINGS DO FOR A LIVING IS THEY CIRCULATE. AND THAT'S TRUE. HOWEVER, ONE DISEASE TENDS TO STAY LOCAL AND YOU CAN ACTUALLY SECURE IT USING LOCAL THERAPY, ALTHOUGH WE DON'T DO THIS ANYMORE IS HODGKINS. AND HODGKINS WAS THE FIRST LYMPHOMA THAT WAS EVER CURED AND THE WORK REALLY CAME FROM USING RADIATION THERAPY. IT WAS SHOWN BACK IN THE LATE 50 MANY TO THE EARLY 60'S THAT HODGKINS TENDS TO SPREAD FROM NODAL GROUP TO NODAL GROUP. SO STAGING CAME ABOUT SIMPLY TO HELP THE RADIATION ONCOLOGIST UNDERSTAND HOW TO RADIATE THEM. AND SO THAT'S REALLY WHERE STAGING CAME FROM. AND IT'S NOW BEEN USED FOR NON-HODGKINS LYMPHOMAS AND IT'S BEEN USED IN A GENERAL WAY WHERE EARLY STAGE DISEASE HAS A BETTER PROGNOSIS THAN LATER STAGE. AS WE REALLY GOTTEN MORE TO UNDERSTANDING LYMPHOMAS, WHAT THE STAGE IS IN TERMS OF THERAPY OTHER THAN MORE LIMITING THERAPY DOESN'T PROBABLY REALLY HELP. SO, WHAT ARE SOME OF THE MAJOR LYMPHOMAS AND WHAT KIND OF OUTCOME DO YOU SEE IN WELL, WE GENERALLY LIKE TO DIVIDE LYMPHOMAS, AND THIS IS REALLY, YOU KNOW, LYMPHOMA 101 INTO WHAT WE CALL END LYNN AND AGGRESSIVE. IN GENERAL, ENDLYNN ARE NOT CURATIVE BUT THEY ARE CALLED ENDLYNN IS BECAUSE THEY GROW SLOWLY AND TEND TO BE CHRONIC DISORDERS AND PEOPLE CAN LIVE WITH THEM FOR MANY YEARS. ON THE OTHER SIDE AGGRESSIVE LYMPHOMAS TEND TO GROW QUICKLY AND KILL YOU QUICKLY BUT THEY TEND TO BE CURATIVE. THAT'S KIND OF THE PARADOX BETWEEN THESE TWO. WOULD YOU RATHER HAVE AN ENDLYNN LYMPHOMA OR AGGRESSIVE. AT THE END OF THE DAY YOU WANT AGGRESSIVE BECAUSE WE CAN CURE MOST OF THEM. IT'S CALLED FOLLICULAR LYMPHOMA AND A LOT OF WORK HAS BEEN DONE ON THIS OVER THE YEARS. THE WHOLE FIELD OF TYPE VACCINE WHICH REALLY I THINK WAS THE PRECURSOR TO USING VACCINES IN SOLID TUMOR WAS FOCUSED ON ENDLYNN LYMPHOMA, FOLLICULAR LYMPHOMA AND WORK HEADED BY JOHN LEVY AT STANFORD. IT'S HAD A LONG HISTORY AND SPUN OFF OTHER THERAPIES THAT HAVE BEEN USEFUL. THESE ARE ALL ENDLYNN LYMPHOMAS THAT YOU GENERALLY MANAGE. NOW THERE ARE SOME PEOPLE OUT THERE WHO THINK WE ARE CURING SOME OF THESE BUT I HEARD THIS MANY TIMES. THE PROBLEM IS WHEN YOU DO A THERAPY ERADICATE IT IN TERMS OF OUR ABILITY TO SEE IT ON A SCAN, YOU ALREADY KNOW IF YOU'RE CURED UNTIL YOU WAIT A VERY LONG TIME BECAUSE YOU CAN KNOCK THE CELL NUMBERS DOWN SO LOW THAT THEY TAKE YEARS AND YEARS AND YEARS FOR THAT TUMOR CELL TO REPRODUCE ENOUGH TO SHOW UP ON YOUR SCAN. SO YOU HAVE TO BE VERY VERY CAUTIOUS. AGGRESSIVE LYMPHOMAS, LARGE CELL LYMPHOMAS, THE MOST COMMON TYPE, HODGKINS DISEASE, THESE TWO ARE BOTH HIGHLY CURATIVE. I WORK ON LARGE CELL, BUT CLASSICAL HODGKINS REALLY IS ANOTHER AREA THAT ARE CURATIVE. MANTEL CELL IS ODD SOME CAN BE ENDLYNN AND NASTY. A LOT OF PEOPLE HAVE WORKED ON THEM A LOT HAVE SAID YOU CAN CURE THEM. MOST OF THEM YOU PROBABLY CAN'T CURE AND WE ARE GOING TO REALLY NEED MORE TARGET THERAPY IN ORDER TO I THINK REALLY MAKE A MAJOR HIT ON A DISEASE LIKE MANTLE CELL LYMPHOMA. NOW, LET'S JUST PREVIOUSLY TALK ABOUT FOLLICULAR LYMPHOMA BECAUSE IT IS THE MOST COMMON TYPE OF LOW GRADE LYMPHOMA. AND YOU CAN SEE HERE IT IS CALLED A FOLLICULAR LYMPHOMA BECAUSE YOU HAVE THE EXPANSION OF THE GERMINAL CENTERS. THESE ARE NOT NORMAL GERMINAL CENTERS. WHAT THEY ARE IS THEY ARE EXPANSIONS OF ALL THESE TUMOR CELLS THAT ARE ACTUALLY BCL2 POSITIVE AND IN THE NORMAL GERMINAL CENTER THE B CELLS DO NOT EXPRESS BCL2 BECAUSE THEY ARE TO UNDERGO APOPTOSIS IF THEY DON'T MEET AN ANTIGEN. SO THE REASON WHY THESE HAVE BCL2, THESE TUMORS DO BECAUSE THEY HAVE A TRANSLOCATION OF THE 14-18 WHICH IS PUTTING THE BCL2 GENE NEXT TO THE PROMOTER FOR ONE OF THE IMMUNOGLOBULINS. AND THIS IS WHAT THE CELLS LOOK LIKE IN THE OLD, THEY USE TO BE CALLED SMALL CLEAVE. THESE ARE ADVANCED STAGE DISEASE, IT DOESN'T SEEM TO MATTER ALTHOUGH PEOPLE WITH EARLY DISEASE LIVE LONGER THAN PEOPLE WITH ADVANCED STAGE, BUT STAGE ONE ONLY COMPRISES AROUND 10% OF ALL CASES. MEDIAN SURVIVAL USED TO BE TEN YEARS. IT'S NOW BEEN PUSHED UP CLOSER TO 15 YEARS BECAUSE OF THE INTRODUCTION OF THE MONOCLONAL ANTIBODY TUSKMATH. IF YOU DIDN'T HAVE SYMPTOMS, OVERSTUDIES SHOWED IT DIDN'T DO ANY GOOD GIVE THEM CHEMOTHERAPY EVEN IF IT WAS AGGRESSIVE CHEMOTHERAPY BECAUSE THOSE PEOPLE WHO GOT CHEMO EARLY VERSUS WHEN THEY NEEDED IT HAVE THE EXACT SAME SURVIVAL. IT'S STILL THAT, IT'S STILL THE SAME ALTHOUGH MORE AND MORE PEOPLE ARE BEGINNING TO WANT TO TREAT THESE TUMORS EARLIER ALTHOUGH THEY'VE NOT PROVEN THAT EARLY TREATMENT HELPS. CHEMOTHERAPY IS OFTEN THE MAIN STAY RADIATION, AND TARGETED AGENTS AND BIOLOGICS HAVE REALLY BURST ON THE SCENE AND I THINK WE'RE ONLY AT THE TIP OF THE ICEBERG IN TERMS OF EFFECTIVE THERAPIES FOR THIS. SO THIS ACTUALLY GIVES YOU WHAT'S CALLED A KAPLAN MAYOR CURVE FOR FOLLICULAR LYMPHOMA RUNNING FROM TO 96 AND YOU CAN SEE HERE LOOKING AT THE PROBABILITY OF PEOPLE WHO ARE LIVING OVER TIME, 50%, AND BANG IT'S AROUND 10 YEARS. FOR 30 YEARS IT DID NOT MOVE ONE BIT. SINCE THEN, SINCE THE INTRODUCTION OF THE MONOCLONAL ANTIBODY WAS APPROVED IN 1998, THESE CURVES ARE NOW LOOKING A LOT LIKE THAT. SO WE ARE SEEING A PROLONGATION IN PEOPLE'S SURVIVAL AS A RESULT OF THE MONOCLONAL ANTIBODY AGAINST CD20. HOWEVER, THE CURVE STILL CURVES DOWN. THEY CHANGE THEIR SLOPE. THAT'S WHY WE DO NOT THINK IN GENERAL WE HAVE BEEN ABLE TO CURE THIS BUT WE CERTAINLY HAVE ALTERED ITS SURVIVAL NATURAL HISTORY. TUSKMAB WAS ARGUABLY THE MOST IMPORTANT DRUG IN LYMPHOMAS IN THE LAST 20 YEARS. IT'S A MONOCLONAL ANTIBODY, THE FIRST ONE ANYBODY EVER APPROVED FOR ANY CANCER. IT WAS APPROVED IN 1998 AND IT WAS APPROVED FOR RELAPSE OR FACTORY LOW GRADE LYMPHOMA SUCH AS FOLLICULAR LYMPHOMA. ITS USING EXPANDED ACROSS ALL OF B CELL LYMPHOMAS. IT HAS EXPANDED INTO AUTO IMMUNE DISEASE, ITP, YOU NAME IT. ANYTHING WHERE THERE SEEMS TO BE AN IMIMMUNOLOGIC UNDERPINNING, RHEUMATOID ARTHRITIS, ETCETERA. A VERY IMPORTANT DRUG, A DRUG WITH LITTLE RELATIVE SIDE EFFECTS. IT IS VERY GOOD AT WIPING ALL OF YOUR B CELLS OUT OF YOUR PERIPHERAL BLOOD. IT DOES THIS AFTER A COURSE OF FOUR DOSES ALTHOUGH IT DOES NOT WIPE OUGHT THE ENTIRE RESERVOIR OF B CELLS BECAUSE THE B CELLS DO COME BACK. WE KNOW THOUGH THAT AS PEOPLE ARE GETTING RITUXIMAB THAT THEY DO SEEM TO BE GOING DOWN SO AS YOU HIT THE NORMAL B CELLS, WE'RE PROBABLY ULTIMATELY AFFECTING THE POOL OF THOSE THAT ARE MORE PLASMA CELL-LIKE BECAUSE THE IMMUNOGLOBULINS CELLS ARE -- THIS IS LOOKING AT RELATIVE CD20 EXPRESSION USING FLOW, USING FLORESCENCE AND YOU CAN SEE THE DISEASE SUCH AS A FOLLICULAR LYMPHOMA, LARGE CELL LYMPHOMA HAVE VERY HIGH LEVELS OF CD20 ON THEIR SURFACE. THESE ARE ALL B CELL LYMPHOMAS AND EVERY SINGLE ONE OF THEM HAS LEVELS OF IT. EVEN A DISEASE LIKE CLL WHERE THE LEVELS ARE RELATIVELY LOW COMPARED TO DISEASE LIKE -- WHERE THE LEVELS ARE IS HE VERY HIGH, IT IS STILL VERY SENSITIVE TO RITUXIMAB BETWEEN GIVEN PROPERLY WITH THERAPY. AND SO THE VERY EARLY STUDIES OF THIS REALLY CAME FROM ITS USE IN FOLLICULAR LYMPHOMA BACK IN 1999 AND 2000 LOOKING AT PEOPLE THAT HAD UNTREATED FOLLICULAR LYMPHOMA AND THEY WERE SEEING RESPONSE RATES THAT WERE IN THE RANGE OF AROUND 70-75% AT A SINGLE AGENT. THIS WAS UNPRECEDENTED. HERE IT IS A BIOLOGIC AGENT BEING ABLE TO HAVE AT LEAST A 50% SHRINKAGE IN AROUND 75% OF PEOPLE WITH LYMPHOMA. AND SO IT WAS THAT KIND OF EVIDENCE THAT I THINK REALLY GOT PEOPLE ACTIVATED WITH THIS. AND AGAIN NOW THIS DRUG IS USED WIDELY AND THAT'S NOT REALLY GOING TO BE THE FOCUS OF THE REST OF THIS LECTURE. SO WHAT ABOUT LARGE CELL? THIS IS THE MODEL THAT I'M REALLY GOING TO SPEND MOST OF THE TALK ON AND REALLY GO OVER SOME OF THE VERY EXCITING SCIENCE THAT REALLY HAS EMERGED FOR THIS. ALREADY MENTIONED TO YOU, THIS IS THE MOST COMMON TYPE OF LYMPHOMA OUT THERE. IT IS ALSO ONE THAT CAN BE CURED. WHY IS IT CALLED LARGE CELL? BECAUSE THE CELLS WERE LARGE, AND THIS OF COURSE WAS NAMED BACK IN THE 1980'S, ACTUALLY USED TO BE CALLED -- BACK IN THE PRE MONOCLONAL DAYS. IT'S LARGER THAN A NORMAL B CELL. >> [INDISCERNIBLE] >> PARDON? >> [INDISCERNIBLE] >> THEY'RE ABOUT TWICE THE SIZE OF A NORMAL LYMPHOCYTE. I DON'T KNOW. I COULDN'T TELL YOU WHAT THE ACTUAL TIME IS. SO THERE'S BEEN A LOT OF WORK LOOKING AT VARIOUS CHEMOTHERAPY REGIMENS IN LARGE CELL LYMPHOMA. BELIEVE IT OR NOT THE VERY FIRST CURATIVE THERAPY FOR LARGE CELL WAS A COMBINATION OF CELLS CALLED CHOP. THIS STANDS FOR THREE DIFFERENT CYTOTOXICS. P STANDS FOR PREDNISONE. THIS REGIMEN CURED ABOUT A THIRD OF LARGE CELLS, AND CHOP CAME FROM THE EARLIER 1970'S. OVER THE NEXT 20 YEARS PEOPLE BEGAN TO KEEP ON ADDING DRUGS ON TOP OF CHOP BECAUSE THIS WAS IN THE DAYS OF WHERE IT WAS UNDERSTOOD THAT COMBINING MULTIPLE CYTOTOXIC CHEMOTHERAPIES SEEMED TO IMPROVE OUTCOME. THEY ARE ACTUALLY ALL CHOP CORES WITH MORE DRUGS PLACED ON TOP. A LOT OF SINGLE INSTITUTIONS WERE PUBLISHING RESULTS LOOKING NICER AND BETTER. OF COURSE AT THE SAME TIME THEY WERE COMPARING THIS TO HISTORICAL DATA WITH CHOP AND THE OUR ABILITY TO DIAGNOSE LARGE CELL WAS BECOMING MORE ACCURATE, SUPPORTIVE CARE WAS BECOMING MORE ACCURATE. AND SO THEY DID A BIG RANDOMIZED STUDY BACK IN THE EARLY 1990'S AND ASKED A QUESTION, IF WE MATCH ALL OF THESE NEW AND IMPROVED VARIATIONS OF CHOP ON CHOP, HAVE THEY IMPROVED OUTCOME. THE ANSWER IS NO. AND SO THIS IS ACTUALLY SOMETHING THAT A GUY NAMED DR. RICHARD FISHER DID AND HE BECAME VERY VERY FAMOUS. AND SOMEBODY WHO WAS EQUALLY FAMOUS ONCE SAID THAT DR. FISHER DID MORE TO UNDERMINE THE TREATMENT OF LARGE CELL THAN ANYBODY ELSE. THAT'S TRUE BECAUSE AS A RESULT OF THIS ANYBODY THAT CAME FORWARD WITH ANYTHING THAT LOOKED LIKE IT WAS BETTER, PEOPLE SAID NO, IT WAS JUST THE SAME, IT'S JUST BECAUSE YOU'RE A SINGLE INSTITUTION STUDIES. SO THIS HAS BEEN A BIG UPHILL BATTLE, BUT THIS IS A DISEASE WHERE I THINK WE'VE MADE MAJOR ADVANCES. SO, HOW DO WE BEGIN TO TRY TO DISSECT DOWN LARGE CELL LYMPHOMA. IT IS NOT IN FACT A SINGLE DISEASE. IN FACT, IT CLEARLY BEHAVES IN A VERY VARIED MANNER. IF YOU'RE ONLY CURING A THIRD OF PEOPLE WITH YOUR CHOP THERAPY, THE TWO THIRDS THAT ARE NOT BEING CURED, IS IT BECAUSE IT'S A DIFFERENT TYPE OF LYMPHOMA, IS IT BECAUSE IT HAS MORE DISEASE, WHAT IS IT? PEOPLE TRY TO DISSECT THIS ALL OUT USING CLINICAL THINGS. AND YOU KNOW, YOU CAN ONLY GO SO FAR WITH CLINICAL THINGS LIKE WHAT STAGE IS IT, WHAT'S THE CONDITION OF A PATIENT, HOW BAD ARE THEIR LAB VALUES. THEY DON'T TELL YOU ANYTHING ABOUT THE MOLECULAR BIOLOGY OF THE TUMOR AND A LOT OF THE DRIVER EVENTS THAT ARE MAKING THAT CELL ACT LIKE IT DOES. AND SO REALLY THIS IS WHERE BACK IN THE REALLY LATE 1990'S -- DEVELOPED A LYMPHOCHIP. HE TOOK CDNA'S THAT HE TOOK FROM B CELL LYMPHOMA CELL LINES, FROM NORMAL B CELLS ETCETERA AND HE CREATED A HOMEMADE LYMPHOCHIP AND HE SAID OKAY, USING GENE EXPRESSION PROFILING, CAN I IDENTIFY LARGE CELL LYMPHOMA AS PERHAPS NOT BEING A SINGLE UNIFIED DISEASE. AND THIS WAS REALLY THE BEGINNING OF THE MOLECULAR PROBING USING GENE EXPRESSION PROFILING OF LYMPHOMAS. AND SPECIFICALLY LARGE CELL LYMPHOMA THAT THEN LED TO IDENTIFY MANY OF THE MUTATIONS THAT ACTUALLY DERIVE IT. AND SO THIS IS ACTUALLY THE VERY FIRST STUDY THAT LOU'S GROUP PUBLISHED USING THE LYMPHOCHIP. THIS HOMEMADE THING. THEY HAVE A MATRIX FULL OF GENOME CHIP THEY DON'T REALIZE BACK IN 2000 YOU ACTUALLY HAD TO MAKE YOUR OWN CHIP. WHAT THEY DID WAS THEY TOOK 274 LARGE CELL SAMPLES. THEY THREW IT ON THIS HOMEMADE CHIP. THEY WENT AHEAD AND LOOKED AT IT TO SEE WHETHER OR NOT THEY COULD IDENTIFY SIGNATURES ASSOCIATED WITH DIFFERENT TYPES. WHAT THEY ACTUALLY FOUND WAS THAT YOU COULD BREAK LARGE CELL LYMPHOMA DOWN INTO TWO MAJOR GROUPS. ONE GROUP THEY CALLED A GERMINAL CENTER LIKE LARGE CELL LYMPHOMA AND THE OTHER GROUP THEY CALLED IT AN ACTIVATED B CELL LARGE CELL LYMPHOMA. WHY DID THEY DO THIS? WHAT DID THEY DO IN ORDER TO LET THEM IDENTIFY WHAT THESE GENES MEANT. AS PART OF THESE ARRAYS, THEY ALSO THREW ON GERMINAL CENTER B CELLS AND POST GERMINAL CENTER OR ACTIVATED B CELLS THAT HAD GONE THROUGH THE GERMINAL CENTER REACTION. IF YOU LOOK AT THE NORMAL GERMINAL CENTER B CELLS EVERYTHING THESE ARE THE SAME GENES HERE. YOU CAN SEE, IT'S NOT EXACTLY THE SAME GENES BUT WHAT IT TURNS OUT IS THAT THESE GENES HERE THAT ARE UP, THESE ARE GERMINAL CENTER GENES, CB10, BCL6, MIC, ETCETERA. THESE ARE NORMAL GENES THAT ARE UPREGULATED WITH AN A NORMAL REGULAR CENTER B CELL AND ON THE OTHER SIDE OF THIS, GENES THAT WERE UPREGULATED IN A NORMAL ACTIVATED B CELL SUCH AS -- PI, IR4, ETCETERA. WAS UPREGULATED IN THESE SO-CALLED ACTIVATED B CELLS. THAT'S WHERE THE NAME CAME FROM AND WHERE THE FIRST MOLECULAR UNDERSTANDING THAT LARGE CELL LYMPHOMA COULD BE BROKEN DOWN INTO TWO LARGE GROUPS. THIS IS ONLY THE BEGINNING OF REALLY TRYING TO UNDERSTAND WHAT'S DRIVING THESE. AND SO IF YOU THEN GO BACK AND LOOK AT WELL, DO WE NORMALLY SEE A NORMAL GERMINAL CENTER B CELL, WELL THE TRANSCRIPTION FACTOR THAT IS NOTABLY INCREASED IN A GERMINAL CENTER B CELL IS BCL6. AND WITHIN AN ACTIVATED B CELL, WHAT IS THE TRANSCRIPTION FACTOR THAT IS NOTABLY TURNED OFF. IT IS AN IO4 AND PLAYS A VERY IMPORTANT ROLE IN INHIBITING BCL6 AND TURN OFF NF KAPPA B. THESE ARE TRANSCRIPTION FACTORS ASSOCIATED WITH THESE NORMAL CELLS AND IT TURNS OUT THEY ARE ALSO KEY TRANSCRIPTION FACTORS THAT ARE ASSOCIATED WITH THESE VARIOUS LYMPHOMA SUBTYPES. AND SO ONCE YOU HAVE A GERMINAL CENTER, YOU CAN IDENTIFY GERMINAL CENTER LARGE CELL OR AN ACTIVATED B CELL LARGE CELL HAPPENS TO BE A THIRD ONE LOU AND I WORK ON EXTENSIVELY BUT I'M NOT GOING TO TALK ABOUT IT TODAY, IT'S CALLED A PRIMARY MEDIAL SPINAL, IF WE LOOK AT THE ORIGIN EACH ONE OF THESE GERMINAL B CELLS, POST GERMINAL CENTER ARE ACTIVATED B CELL FOR THE ABC TYPE AND A B CELL FOR THE PRIMARY MEDIASTINUM TYPES. YOU ONLY FIND TRANSLOCATION OF BCL2 AND C WELL AM BLAH CASE IN THE GERMINAL CENTER TYPE. YOU ONLY FIND ACTIVATION OF NF KAPPA B IN THE ABC TYPE AND YOU ONLY FIND AMPLIFICATION OF PDL1 AND 2 ON CHROMOSOME 9, P24 THAT MEDIASTINUM TYPES. UNIQUE ONCOGENIC EVENTS ASSOCIATED WITH EACH OF THESE TYPES THAT WERE INITIALLY IDENTIFIED USING GENE ARRAY. NOW THE NEXT QUESTION YOU WANT TO ASK IS IF THESE ARE IN FACT DIFFERENT TYPES OF LYMPHOMAS, DO THEY HAVE DIFFERENT OUTCOMES. AND THE ANSWER IS YES. IF YOU LOOK AT R CHOP WHICH IS NOW CHOPPED PLUS RITUXIMAB STANDARD OF CARE IS THERE A DIFFERENCE OF OUTCOME BETWEEN THOSE TUMOR TYPES OF THE GERMINAL CENTER TYPES VERSUS THE ACTIVATED B CELL TYPE THIS IS LOOKING AT OVERALL SO-CALLED PROGRESSION-FREE SURVIVAL MEANING THAT THE DISEASE STAYS IN REMISSION OR OVERALL SURVIVAL. WHAT YOU SEE HERE IS THAT THOSE TUMORS THAT ARE OF THE GERMINAL CENTER TYPE HAS A BETTER OUTCOME THAN THOSE TUMORS OF THE ACTIVATED B CELL TYPE. AND SO ONE OF THE QUESTIONS IS WHY. WELL THIS WAS ANOTHER LARGE ANALYSIS WHERE LOU'S GROUP LOOKED AT GENE ARRAY OF A LARGE NUMBER OF SAMPLES ARE R CHOPPED BASE THERAPY. THE INITIAL ANALYSIS I SHOWED YOU FROM 2000 WAS IN CHOPPED BASE THERAPY BECAUSE WHERE RITUXIMAB HAS NOT BEEN SHOWN, IT WAS NOT USED YET BECAUSE IT WAS JUST APPROVED IN 1998, IT TURNS OUT RITUXIMAB INCREASES CURE RATE BY 8%. THEY LOOKED AT VARIOUS INDICES OR SIGNATURES THAT MIGHT HELP PREDICT OUTCOME AND ACTUALLY WHAT THEY FOUND WAS THAT THE MICRO ENVIRONMENT SEEMED TO PLAY A ROLE AS WELL SUCH THAT THOSE TUMORS THAT HAD A STROMA ONE SIGNATURE HAD AN IMPROVED OUTCOME WHEREAS THOSE TUMORS THAT HAD THE SO-CALLED STROMAL TWO SIGNATURE HAD A WORSE OUTCOME. IT'S NOT ONLY THE TUMOR CELLS ITSELF BUT IT'S ALSO RELATED TO THE MICRO ENVIRONMENT AS WELL. AND YOU COULD ACTUALLY SEE THAT THE STROMAL 1 SIGNATURE IS SEEN ACROSS THE GERMINAL CENTER TYPES AND STROMAL 2 IS SEEN ACROSS BOTH TYPES. SO UNLIKE THESE OTHER ONCOGENIC EVENTS SUCH AS CONSTRICTED ACTIVATION OF NF KAPPA B OR BCL6 THAT APPEARED TO BE RESTRICTED TO DIFFERENT SUBTYPES, YOU CAN SEE THESE MICRO ENVIRONMENT EVENTS ACTUALLY ARE SEEN ACROSS THESE DIFFERENT TYPES. AND YOU CAN ACTUALLY COME UP WITH A SCORE, A PREDICTIVE SCORE JUST BASED ON SOME OF THESE MICRO ENVIRONMENT EVENTS. SO HOW MIGHT WE BEGIN TO THINK ABOUT USING SOME OF THIS NEW KNOWLEDGE ABOUT GERMINAL CENTER VERSUS ABC AND TRYING TO UNDERSTAND HOW WE MIGHT IMPROVE THERAPY. WELL, IT WAS THROUGH THE RECOGNITION THAT THERE'S A SPECIFIC TYPE OF LARGE CELL LYMPHOMA THAT IS DERIVED FROM A GERMINAL CENTER CELL THAT WE BEGIN TO REALLY UNDERSTAND THAT BCL6 MAY BE PLAYING AN IMPORTANT ROLE. IN FACT THE NORMAL FUNCTION OF BCL6 IS TO SET YOUR NORMAL B CELL UP TO BE RELATIVELY RESISTENT TO APOPTOSIS, AND TO BE PROLIFERATED. BECAUSE THAT NORMAL B CELL IS RANDOMLY MUTATED SEMANTICALLY MUTATING ITS IMMUNOGLOBULIN GENES SO THAT YOU HAVE ALL THESE DIFFERENT VARIATIONS IN THE HOPE THAT ONE OF THEM WILL FIT AN ANTIGEN THAT'S FLOWING THROUGH THE GERMINAL CENTER. SO WHEN A NORMAL B CELL IS MUTATING SPONTANEOUSLY, IT'S IMMUNOGLOBULIN GENES THAT'S ACTUALLY VIEWED BY THE CELL AS A STRESS EVENT AND THAT CELL WITH UNDERGO APOPTOSIS. OF COURSE THE REGULATOR OF THIS IS THE P53 AXIS. IN ORDER TO KEEP THESE NORMAL B CELLS FROM JUST ALL CROAKING AS THEY ARE UNDER GOING THESE SPONTANEOUS SEMANTIC HYPER MUTATIONS, BCL6 INHIBITS P35. IT ALSO INHIBITS REGULATORS OF PRO-LIFE RATIONS SUCH AS P21 AND P27 SO THAT THESE NORMAL B CELLS ARE ABLE TO REPLICATE THEMSELVES. SO BASICALLY THE NORMAL FUNCTION OF BCL6 IS TO REDUCE THE DAMAGE RESPONSE AND INCREASE NORMAL B CELL PROLIFERATION. THAT'S KIND OF LIKE THE PERFECT STORM FOR A LYMPHOMA BECAUSE IT WANTS TO NOT BE SENSITIVE TO GENO TOXIC STRESS BECAUSE THE TYPES OF DRUGS WE USE INDUCE GENO TOXIC STRESS AND MANY OF THEM KILL BY ACTIVATING THE P53 AXIS WITHIN THE NORMAL, WITHIN THE LYMPHOMAS. SO BCL6 ACTUALLY DIALS ALL THIS DOWN NORMALLY AND ALSO ALLOWS THE TUMOR CELL TO REP INDICATE AS WELL. SO VERY LOGICAL TARGET, IF YOU WANTED TO GO AFTER THE GERMINAL CENTER WOULD BE BCL6. WE HAVE NOT FOUND A GOOD TARGET FOR IT. PEOPLE ARE USING EPI GENETIC MODULATORS TO SCAN REGULATE IT. SOME ARE TRYING TO INHIBIT BCL6 AS A TRANSCRIPTION FACTOR BUT GENERALLY TARGETING TRANSCRIPTION FACTORS ARE CLINICALLY HAS BEEN RELATIVELY DIFFICULT AND THAT'S BEEN ACROSS OTHER ONES AS WELL. WE DON'T HAVE ONE FOR NF KAPPA B, WE DON'T HAVE ONE FOR MIC, ETCETERA. WELL, THIS ACTUALLY IS JUST LOOKING AT BCL6 EXPRESSION. YOU CAN SEE THAT IT REALLY IS, I MISSPOKE EARLIER BY THE WAY. TRANSLOCATIONS OF BCL6 ARE ONLY FOUND WITHIN THE GERMINAL CENTER BUT THE OVER EXPRESSION OF BCL6 CAN ACTUALLY BE FOUND IN SOME OF THE ABC TYPES. AND WHAT HAPPENS IS THAT MOST OF THEM HAVE DOWN REGULATION OF BCL6 BECAUSE AS BCL6 GOES DOWN NF KAPPA B GOES UP AND YOU CAN SEE MOST OF THE GERMINAL CENTERS HAVE RELATIVELY HIGH BCL6. SO IT IS FUNCTIONING TO INHIBIT APOPTOSIS AND TO PROMOTE PROLIFERATION. HAVEN'T BEEN ANY GOOD TARGETS BUT IT ACTUALLY TURNS OUT THAT IF YOU USE A DRUG LIKE -- IT'S LOOKING AT A SINGLE CONCENTRATION AND LOOKING AT OVER TIME SIMPLY EXPOSING A TUMOR CELL TO E TOP SIDE YOU CAN SEE BCL6 IS DOWN REGULATED. THIS IS PROBABLY A NORMAL EXPAWNS BECAUSE AGAIN A JEANNIE TOPIC SUPPRESS COMES IN AND IT WANTS TO TURN OFF THE INHA BUSINESS OF P53. THIS IS A DOSE DEPENDENT ZERO VERSUS 20 AND YOU CAN SEE IT ACROSS A VARIETY OF DIFFERENT LINES. AND IF YOU ACTUALLY LOOK AT THE CONS LATIONS HERE AND LOOK AT BCL LEVELS HERE AND INDICES OF DNA BREAKS AS YOU EXPOSE THE CELL TO MORE TOPSIDE YOU INDUCE APOPTOSIS. SOCIETY IT LOOKS LIKE OTHER SUMMARY INHIBITORS THAT BRING HIGH GENO TOXIC STRESS ARE VERY GOOD WAYS TO GET AT BCL6. AND WE ACTUALLY HAVE BEEN WORKING ON MANY YEARS HERE ON SOMETHING CALLED E POCK R WHICH MADE IT WAY INTO BING STANDARD FOR LARGE CELLS. BUT LOOKING AT IT WITHIN GCB TIN, IT USES TWO VERY POETANT SUMMARIES, TWO INHIBITORS AND TOPSITE -- AND IT'S VERY HIGHLY GENO TOXIC STRESSOR. AND ACTUALLY IF YOU LOOK AT THE OUTCOME OF LARGE CELL LYMPHOMA, I SHOWED YOU WITH CHOP ALONE, THE CURE RATE WAS AROUND 32%. WHERE YOU ADDED RITUXIMAB AFTER THAT THE CURE RATE WENT UP TO 60%. THIS IS A MULTICENTER STUDY SHOWING THAT WHEN YOU USE DOSE DOSE EPOCH R THE DOSE RIGHT MOVES UP TO AROUND 81%. IF YOU LOOK AT THIS IN THE GERMINAL CENTER GROUP, 100% OF THE GERMINAL PATIENTS HERE WERE WITHOUT DISEASE PROGRESSION. SO THE EPOCH SIDE AND ALL OF THE -- MAY BE VERY EFFECTIVE WITHIN THIS REGIMEN BECAUSE IT IS ADDRESSING THE ISSUE WITH THE BCL6 TRANSCRIPTION FACTOR. THERE'S NO OTHER INHIBITORS OUT THERE YET SO THIS IS THE BEST WE'VE COME UP WITH. THERE'S NOW A LARGE RANDOMIZED STUDY. SO THE OTHER BIG AREA THAT WE WORKED ON IS TARGETING THE ABC SUBTYPE. THIS HAS REALLY BEEN A FASCINATING STORY THAT LOU HAS REALLY UNWOUND. AND THIS IS ACTUALLY LOOKING AGAIN AT THE GENE EXPRESSION PROFILING, LOOKING AT THE ACTIVATED B CELL TYPE HERE. AND THESE ARE LOOKING AT NF KAPPA B TARGET GENES. SO ONE OF THE THINGS THAT LOU NOTED WHEN HE DID THE ORIGINAL ARRAY BACK IN 2000 WAS THAT CONSTITUENT ACTIVATION OF NF KAPPA B WAS A MAJOR FACTOR IN THE ACTIVATED B CELL TYPE. AND ALL OF YOU KNOW THE TARGETS OF THIS TRANSCRIPTION FACTOR ARE TO TURN UP MIC. AND THEY TURN UP A VARIETY OF ANTI-APOPTOSIS GENES. THEY TURN OFF PROLIFERATION GENES, ETCETERA. SO NF KAPPA B IS DRIVING ALSO A LOT OF SIMILAR EVENTS AS BCL6 IN A VERY DIFFERENT WAY. AND SO WE ACTUALLY PERFORMED A CLINICAL EXPERIMENT ABOUT, GOODNESS, WE PUBLISHED AT ABOUT THREE YEARS AGO NOW AND STARTED AT ABOUT TEN YEARS AGO. AND ONE OF THE QUESTIONS WE ASKED WAS IF WE COULD CLINICALLY SOMEHOW USE A STRATEGY TO INHIBIT NF KAPPA B, COULD WE IMPROVE THE OUTCOME OF ABC LARGE CELL. THE IDEA BEING THAT IF YOU DOWN REGULATE NF KAPPA B, THE CELL BECOMES MORE SUSCEPTIBLE TO YOUR GENO TOXIC STRESSORS AND THEREFORE OUTCOME WOULD BE IMPROVED. WHY DID WE WANT TO DO THIS? WE DID THIS BASED ON AN EXPERIMENT, LOU DID IN THE LABORATORY, WHERE HE TOOK AN INHIBITOR OF I KAPPA B KINASE WHICH PREVENTS THE PHOSPHORYLATION OF I KAPPA B AND IT PHASE WITHIN I KAPPA B AND THE CYTOPLASM -- THEN IT IS TARGETED FOR -- LEAVING NF KAPPA B FREE TO GO OFF INTO THE NUCLEUS. AND WHEN HE EXPOSED ABC CELL LINES TO THIS I KAPPA B KINASE INHIBITOR IT KILLED THEM BUT IT DIDN'T DO ANYTHING TO THE GERMINAL CENTERS. BECAUSE THEY DIDN'T HAVE ACTIVATION OF NF KAPPA B. AND SO WE WANTED TO PERFORM A CLINICAL EXPERIMENT. SO THIS IS THE FIRST GENERATION ATTEMPT TO TRY TO TARGET NF KAPPA B IN LARGE CELL. I KAPPA B KINASE AS YOU MIGHT IMAGE TURNS OUT TO BE AN EXTREMELY LETHAL TARGET BECAUSE NF KAPPA B IS INVOLVED IN MANY DIFFERENT CELLS. AND SO IT WAS, SO INHIBITING IT IS NOT A GOOD THING. BUT WE DID HAVE A DRUG THAT HAD JUST RECENTLY BEEN APPROVED THAT ACTUALLY INHIBITS THE DEGRADATION OF THE PHOSPHO I KAPPA B. IF YOU EXPOSE TUMOR CELLS TO -- YOU ACTUALLY CAN INHIBIT NF KAPPA B. SO WE DID A STUDY WHERE WE TOOK RELAPSE LARGE CELL LYMPHOMAS. WE DID A BIOPSY ON THEM. WE EXPOSED THEM -- TO GET A RESPONSE RATE AND IF THEY GREW THROUGH THIS OR WEREN'T WELL ENOUGH, THEY WOULD GET -- PLUS A CHEMOTHERAPY REGIMEN. WE USED EPOCH, AND THEN WE LOOKED TO SEE WHETHER OR NOT THERE WAS A DIFFERENCE IN OUTCOMES BETWEEN THOSE PATIENTS THAT HAD AN ABC AND A GERMINAL CENTER TYPE. NOW, WHAT WE HAD PREVIOUSLY, WHAT WE FOUND WAS THIS. THIS IS THE MEDIAN SURVIVAL OF THE PATIENTS ON THIS CLINICAL TRIAL. AND IT WAS EIGHT MONTHS. THIS IS PRETTY TYPICAL OF WHAT YOU SEE IN RELAPSE REFACTORY OF LARGE CELLS. IF YOU ARE NOT CURED UP FRONT, YOU DIE PRETTY QUICKLY. SO THE FIRST THING WE ACTUALLY ASKED WAS WHETHER OR NOT THERE WAS A DIFFERENCE BETWEEN THE OUTCOME OF ABC AND GCB CELL LYMPHOMA. AS YOU SAW IN A PREVIOUS SLIDE I SHOWED YOU THAT THE ABC TYPE DOES WORSE. SO IF THE ABC TYPE DID BETTER THAN THE GERMINAL CENTER TYPE WITH JUST THE ADDITION OF BORTEZOMIB, THE LIKELIHOOD IS WE WERE MODULATING THE ACTIVITY OF ABC THAT DOES WORSE. IN FACT WHAT WE FOUND WAS THOSE TUMORS THAT MOLECULARLY WERE ABC AND ACTUALLY HAD A WHOPPING 82 RESPONSE RATE WHICH IS UNPRECEDENTED IN RELAPSE CASES WHEREAS THE GERMINAL CENTER GROUP WAS ONLY 13%, AND WE HAD A SIGNIFICANTLY LONGER SURVIVAL IN ABC VERSUS THE GERMINAL CENTER GROUP. SO THIS WAS A FIRST GENERATION ATTEMPT TO TRY TO MODULATE AND LOOKED AS THOUGH IN THIS EXPERIMENT, THAT IS REALLY WHAT THIS WAS, THAT PERHAPS BORTEZOMIB WAS SENSITIVE TO CHEMOTHERAPY AS A RESULT OF THIS, THERE'S NOW A LARGE INTERNATIONAL RANDOMIZED STUDY. NOW, WHAT'S REALLY COME OUT, AND WHAT WE'RE NOW WORKING ON IS THIS WHOLE AREA OF BCR RECEPTOR SIGNALING. WE'RE ON THE VERGE OF ANOTHER MAJOR BREAK THROUGH FROM A DRUG POINT OF VIEW IN TERMS OF THE TREATMENT OF BCL LYMPHOMA. AND IT IS TARGETING THE BCR RECEPTOR. JUST LIKE RITUXIMAB TARGETED CD20 AND BY THE WAY THE MECHANISM OF RITUXIMAB IN TERMS OF HOW IT WORKS IS NOT REALLY BEEN WELL ELUCIDATED. IT PROBABLY WORKS FOR BOTH IMMUNOLOGIC MEANS AND ALSO MODULATING SIGNALING. HOWEVER, THE FOCUS NOW IN LOU'S LAB AND IN MY CLINICAL GROUP HAS BEEN WORKING ON BCR RECEPTOR SIGNALING. SO, THERE IS A NORMAL TYPE OF ACTIVE BCR SIGNALING WHICH IS THROUGH THE CD39A AND B BCR RECEPTOR. WHEN THIS BCR RECEPTOR ENCOUNTERS ANTIGEN, IT SIGNALS DOWN THROUGH FOUR DIFFERENT MAJOR AREAS. OF YOU MAY HAVE HEARD THAT THE SIGNALS DOWN TO KINASE MKTM TOUR. IT SIGNALS DOWN INTO THE -- PATHWAYS AND SIGNALS DOWN THROUGH MASS KINASE AND A MAJOR SIGNALING IS DOWN HERE TO THE NF KAPPA B PATHWAY. AND THAT IS SOMETHING THAT LOU RECOGNIZED. OBVIOUSLY MANY PEOPLE KNEW THIS. SO THE QUESTION IS IF YOU HAVE ACTIVATION OF NF KAPPA B IN ABC, ARE THERE ANY EVENTS UPSTREAM THAT YOU COULD IDENTIFY WITHIN TUMOR CELLS THAT IN FACT MAY BE DRIVING THIS OTHER THAN NORMAL ANTIGEN STIMULATION, WHICH YOU COULDN'T RULE OUT EITHER. AND SO ACTUALLY, THERE'S TWO DIFFERENT KINDS OF SIGNALING. THERE'S THE NORMAL SIGNALING, THERE'S ANOTHER TYPE OF SIGNALING CALLED TONIC BCR SIGNALING THAT WE'RE NOT GOING TO BE DEALING WITH. AND THIS IS A KAPPA SIGNALING THAT SEEMS TO FOCUS MOSTLY ON THE M TOUR. HOWEVER, LOU IDENTIFY WHAT HE CALLS CHRONIC ACTIVE BCR SIGNALING, AND IN FACT IT IS EMERGING THAT THERE'S PROBABLY AN AUTO ANTIGEN THAT IS STIMULATING THE BCR RECEPTOR IN THESE TUMOR CELLS CAUSING THIS KIND OF LOW GRADE CHRONIC ACTIVE BCR SIGNALING THAT IS SIGNALING DOWN AND TURNING ON NF KAPPA B AND LEADING TO A VERY POTENT SURVIVAL SIGNAL FOR THE TUMOR CELL. AND SO HE MARCHED UP THE SERIES OF PROTEINS AND ASKED ARE ANY OF THESE MUTATED. AND IS THERE IS A PROTEIN CALLED CART 11 THAT FORMS A COMPLEX WITH MALT ONE AND BCL2 WHICH IS ESSENTIAL FOR THE ACTIVATION OF THE I KAPPA B KINASE. WHAT HE ACTUALLY FOUND WAS THAT THERE ARE ACTIVATING MUTATIONS IN THE COIL AREA OF CARD 11 IN AROUND 10 TO 12% OF CASES OF THESE ABC LARGE CELLS. ANOTHER IMPORTANT KINASE IS BRUTON TYROSINE KINASE AND THEY DIDN'T FIND ANY MUTATIONS THERE. HOWEVER, THEY ALSO LOOKED AT THE BCR RECEPTOR ITSELF, AND THEY ACTUALLY FOUND MUTATIONS WITHIN THE ICAM MOTIVES -- BUT CD79A AS WELL. AND THESE ALL CONTRIBUTE TO STIMULATING OR AMPLIFYING THE BCR SIGNALING WHICH LEADS TO NF KAPPA B. AND IF YOU ACTUALLY LOOK AT THE VARIOUS, THE RATES OF THE ACTUALLY BCR MUTATIONS, YOU'RE ALMOST EXCLUSIVELY FOUND WITHIN THE ABC TYPE, WITH A VERY SMALL NUMBER FOUND IN GERMINAL CENTER AND NONE FOUND IN BIRKETT OR MALTEN FROM LINES. NOW THERE'S ANOTHER PATHWAY THAT'S INVOLVED HERE AND THAT'S THE TOTAL LIKE RECEPTOR PATHWAY WHERE THROUGH MIGHTY 88 AND IRAC4 AND COOPERATES WITH THE BCR SIGNALING TO A VALUATE I KAPPA B KINASE AND THEY FOUND A HIGH RATE OF MUTATIONS ACTIVATING MUTATIONS IN MIGHTY 88 AS WELL. AND ACTUALLY IF YOU LOOK AT THE VARIOUS OVERLAP OF THE VARIOUS MUTATIONS, THERE'S THE CD79B OR A OR MIGHTY 88 MUTATION IN 42% OF ALL LARGE ABC LARGE CELLS. AND IF YOU LOOK AT MIGHTY 8 OVERALL IT'S 29%, 23% OF CASES. AND SO THESE ARE VERY PREVIOUS LUNTD MUTATIONS WHICH IN AND OF THEMSELVES ARE PROBABLY NOT ENOUGH TO DRIVE THE CELL BUT THEY PROBABLY ACT IN SOME WAY AS AMPLIFIERS OF WHAT WE THINK IS PROBABLY CHRONIC ACTIVE STIMULATION OF THE BCR RECEPTOR. AND SO WE'VE BEEN VERY INTERESTED IN THIS AND SO A VERY INTERESTING DRUG IS A DRUG CALLED -- WHICH IS A HIGHLY SPECIFIC INHIBITOR OF BRUTON TYROSINE KINASE AND IT ACTUALLY FITS INTO THE BINDING POCKET AND IT FORMS A COVALENT BOND WITH A SISTINE. THERE ARE ONLY NINE KINASES WITHIN IN THE GENOME AND THE BINDING POCKET SO THIS IS A HIGHLY SPECIFIC DRUG AND BTK IS ONLY PRESENT IN, ONLY APPEARS TO BE INVOLVED IN THE BCR SIGNALING CASCADE. AND SO WHEN THEY ACTUALLY LOOKED AT IT WITHIN CELL LINES, THEY FOUND THAT IF THERE WAS AN ABC CELL LINE WHERE WE BELIEVE THIS SIGNALING IS GOING ON, THE IBRUTINIB HAD EFFECT WHEREAS IN THE GERMINAL CENTER GROUP THEY DIDN'T SEE. SO WE'VE ACTUALLY STARTED BOTH A PILOT TRIAL AND NOW A PHASE TWO TRIAL THAT'S ACTUALLY DONE WHERE WE LOOKED AT IBRUTINIB IS A SINGLE AGENT IN A LARGE CELL. I'M JUST GOING TO SHOW YOU A COUPLE RESPONSES. THIS IS ACTUALLY A CASE THAT WAS PRIMARY REFRACTORY MEANING THAT NOTHING EVER WORKED. ALL THE CHEMOTHERAPY SHE WAS GIVEN, NOTHING WORKED. AND WITHIN THREE WEEKS SHE HAD MASSIVE DISEASE THAT VIRTUALLY WENT AWAY WITH THIS ORAL DRUG THAT CAUSES VIRTUALLY NO SIDE EFFECTS. AND IF WE ACTUALLY LOOK AT THE TEN CASES WE ACTUALLY TREATED TWO OF THEM WENT INTO A COMPLETE REMISSION. ONE INTO A PARTIAL REMISSION AND ONE OF THE STABLE DISEASES THAT HAD ABOUT A 35% SHRINKAGE WAS GONE ON TO DO A TRANSPLANT AND SEEMS TO BE IN REMISSION AS WELL. WE'VE ALSO NOW DONE, WE'VE NOW COMPLETED A PHASE TWO TRIAL AND THOSE RESULTS WILL BE PRESENTED AT ASH. NOW ONE OF THE QUESTIONS YOU MAY ASK IS, IS THERE A RELATIONSHIP BETWEEN THE VARIOUS MUTATIONS 79B AND MIGHTY 88. AND THERE'S AN EARLY EMERGING PICTURE THAT IF YOU HAVE NO MUTATION OF EITHER THE DRUG WORKS, IF YOU HAVE 79B AND MIGHTY 88, THE DRUG WORKS. BUT IF YOU HAVE MIGHTY 88 ALONE, THE DRUG DOESN'T WORK. MEANING THAT SUGGESTING TO US THAT IF IT'S MIGHTY 88 ALONE, THAT THAT HAS BECOME THE DOMINANT DRIVER AND THEREFORE INHIBITING UPSTREAM OF THE BCR SIGNALING CASCADE WILL NOT BE USEFUL EARLY RESULTS. WHAT DO WE THINK ABOUT TARGETING IN THE GERMINAL CENTER, BCL6 IS REALLY A VERY IMPORTANT TARGET, NON-SPECIFIC TARGETING WITH CHEMOTHERAPY MAY WORK WITHIN THE ACTIVATED B CELL TYPE. NF KAPPA B AND REALLY BCR SIGNALING. THERE'S A VARIETY OF OTHER TARGETS THAT CAN BE LOOKED AT WITHIN THE BCR SIGNALING CASCADE. ALSO INHIBITORS THAT ARE COMING UP ON THE MARKET FOR INVESTIGATION OF IRAC4. I HAVEN'T TALKED MUCH ABOUT M TOUR INHA BUSINESS. IT IS THE DRIVING FORCE FOR LARGE CELLS. WITH THAT CLINICAL TRIALS HAVE BEEN DONE BY MY GROUP. ELAINE AND -- DO ALL THE LABORATORY WORK AND OF COURSE LOU, ALL THE SCIENCE REALLY HAS BEEN DRIVEN BY LOU. THANK YOU. [APPLAUSE] >> WE'RE VERY PLEASED TO HAVE POMMIER AS OUR NEXT LECTURE. MONDAY IS THE COLUMBUS DAY HOLIDAY SO WE HAVE THE TRACO LECTURE ON TUESDAY NEXT WEEK, NOTMONDAY. HE WAS EDUCATED AT THE UNIVERSITY OF PARIS, HE'S NOW CHIEF OF THE LABORATORY OF MOLECULAR PHARMACOLOGY HERE AT CCI >> IT'S GREAT TO -- THE DRUG AND THE INHIBITORS SO WE JUST REVIEW THE MORE BASIC LEVELO THESE DRUGS TOO. SO DNA -- TARGET OF DRUG IN THE CLINIC. BOTH IN CANCER AND BACTERIAL DISEASE, INFECTIOUS DISEASES. SO THIS REVIEW SHOULD BE USEFUL TO YOU IF YOU WANT TO KNOW MORE ABOUT THE DETAIL. A NUMBER OF THE SLIDES ARE PUT INITIALLY -- SLIDE COMES OUT OF THIS REVIEW. I MAY GO QUICKLY AND YOU MAY NOT UNDERSTAND THE DETAILS BUT YOU COULD GO BACK TO THIS AND THEN YOU CAN E-MAIL ME IF YOU WANT TO. THIS IS A BOOK THAT JUST CAME OUT, SO IF YOU GO TO THE LIBRARY, YOU COULD ALSO GO IN THERE AND LOOK FOR YOURSELF, THE FIELD. THERE'S CLEARLY A VERY LARGE AMOUNT OF LITERATURE. TOPOISOMERASES ARE RELATED TO CANCER, NOT ONLY BECAUSE THEY ARE TARGET OF DRUGS BUT BECAUSE THEY ALSO ARE THE INITIATING EVENT IN A NUMBER OF CANCERS. AS WYNDHAM MENTIONED THERE'S LEUKEMIA CONNECTED TO INHIBITORS AND IT'S THOUGHT NOW THAT CHILDREN LEUKEMIA EARLY PHASE COULD BE DUE TO FOOD THAT POISON AND COULD POTENTIALLY GENERATE TRANSLOCATIONS AND RECOMBINATION AND GENERATE THE LEUKEMIA IN THOSE CHILDREN. SO THE TOPOISOMERASE ARE GOOD AND BAD BUT COULD GENERATE CANCER BUT COULD BE USED TO TREAT CANCER. THEY ARE USED TO TREAT CANCERS. THE FAMILY OF TOPOISOMERASES IS LARGE AND IT'S NOT A SINGLE TOPIC, UNFORTUNATELY BUT IT'S NOT MY DOING. THERE ARE SIX GENES IN HUMAN CELLS. THESE GENES ARE DIVIDED IN THREE TYPES WHICH WE THEN REFER TO AS TOP ONE, OR TOPOISOMERASE TOP WHICH IS THE NAME THE GENE IS GIVEN, USUALLY THEY ARE CALLED TOP, TOP 1, TOP 2, TOP 3. IN EACH THERE ARE TWO WHICH IS STRAIGHTFORWARD SO FAR. WHERE IT GETS MORE COMPLICATED IS THAT THERE ARE TWO TYPES WHAT WE CALL TYPE ONE AND TYPE TWO. TYPE ONE ARE ENZYMES THAT IS FROM THE DNA. SO DNA DUPLEX AND FOR TOPOISOMERASE IS TO DO THEIR MAGICAL DNA UNRAVELING PROCESS. SO BASICALLY A TOPOISOMERASE ONE EASY WAY TO THINK OF A TOPOISOMERASE IS TO THINK OF YOUR TELEPHONE CORD OR YOUR COMPUTER CORD ANY TIME YOU PUT THIS IN YOUR BAG IT'S GOING TO BE ALL TWISTED AND TANGLED AND IT'S IMPOSSIBLE TO UNTANGLE IT. SO TOPOISOMERASE IS A SIMPLE SOLUTION. IT'S JUST GOING TO CUT THE BIG ONE AND THEN IT WILL HOLD IT AND JUST PASS IT THROUGH AND LIGATE IT GIVEN. SO IT'S MAGICAL. IT EITHER CUTS ONE STAND ONLY OR TWO STRANDS. WHEN ONE STRAND IS TYPE ONE AND WHEN YOU CUT TWO STRANDS IT'S TYPE TWO. SO TWO FOR TWO STRANDS, ONE FOR ONE STRAND. AND THEN THERE ARE TWO SUBGROUPS, THE TYPE ONE A WHICH IS ACTUALLY TOP 3 AND THEN TYPE 1B WHICH IS TOP 1. THE REASON THEY ARE CALLED A AND B IS BY THE CHRONOLOGY OF DISCOVERY. TYPE ONE WAS FIRST DISCOVERED IN BACTERIA. AND THEN TYPE 1B WAS DISCOVERED LAST. IN ANY CASE ALL THE HISTORY COULD BE LOOKED AT IF YOU'RE REALLY CAREFUL IN THE FIRST CHAPTER OF THIS BOOK. THE CLOSING ACTIVITY, BREAKING DNA, AS I SAID FOR TOP 1 YOU'RE BREAKING ONE STRAND AT A TIME -- AND THEY ARE USUALLY PRETTY LARGE. ANY OF THESE AS A UNIT IS -- LARGE PROTEIN. AND TOP 2 BINDS, YOU COULD ALSO ASSOCIATE THESE TO MEMORIZE, TOP 2 IS DIMER. THIS IS DIMER. AND THEN YOU HAVE A HUGE MACHINE DOWN TO THE DNA. THAT'S MUCH BIGGER THAN A NUCLEOSOME -- AND IT'S WORTHWHILE IN TERMS OF MEDICAL APPLICATION. THE LANDSCAPE OF TOPOISOMERASE IS IN HUMANS, IN A HUMAN CELL VERSUS BACTERIAL CELL. YOU COULD FIRST SEE THAT THERE'S AN ENRICHMENT IN THE HUMAN CELLS. THERE ARE TOPOISOMERASE IN THE HUMAN CELLS. YOU HAVE TWO TYPE 1B YOU HAVE TWO TYPE 2A. THESE AGAIN ARE THE GENES THAT ARE MENTIONED BEFORE. ONE AND -- E COLI DOESN'T HAVE 1B. SO E COLI LIVES ON 1A. BUT 1A HAS TWO FORMS. E COLI WOULD TEND TO BE TOP. SO IT'S TOP 1 AND TOP 3. THE BACTERIA, THERE'S ACTUALLY MORE DIVERGENCE -- DISCOVERED HERE AT NIH -- OVER AT NASH. AND THEN TOP 4 IS FOR -- THE K IS SOMEWHAT SIMILAR BUT RICHER IN THE TOP 2 PART TO SEPARATE THE CHROMOSOMES. THE BIOCHEMISTRY OF TOPOISOMERASE IS COMMON. AND WHEN THEY DO ALL THE TOPOISOMERASES DO THE SAME. THEY USE A TYROSINE IN THE ENZYME TO ATTACK THE DNA DIE STROME. AND THE TYROSINE IS VERY ACTIVE SO ALL IT TAKE IS FOR THE ENZYME TO BIND TIGHTLY, BRING THE TYROSINE IN THE RIGHT ORIENTATION -- AS A RESULT OF WHICH YOU GET A BREAK WITH THE COVALENCE OF THE OWN SUOMI -- ENZYME TO THE OTHER SIDE. SO TOP 1 WOULD LEAD TO THE -- HYDROXEN AND THIS REACTION IS COMPLETELY ARE YOU VERSABLE. THAT'S WHAT I MEANT WHEN I SAID PLEASE HOLD FROM THE DNA'S OWN, IF IT'S ALL RELAXED AND IT RUNS TOGETHER AND THEN THIS WILL THEN ATTACK BACK AND REVERSAL REACTION. IN THE CASE OF TOP 2, THE POLARITIES IS -- SO TOP 2 AND TOP 3 ALWAYS LEAD TO THE FACT -- WHY DOES IT MATTER. IT DOESN'T MATTER BECAUSE TRIPS BACTERIA AS I SAID ONLY USE THIS TYPE OF LINKAGE. ON THE OTHER HAND -- THE VIRUSES ON HUMANS USE THIS TOP 1 VERY HEAVILY. AND TOP 1 THEN IS PART OF A FAMILY WHICH WE CALL THE TYROSINE, TYROSINE RECOMBINASE FAMILY. SOME OF YOU MAY KNOW FOR THIS IS THE RECOMBIN ACE. IT SEEMS TO RETURN FROM THE -- THESE ENZYMES MOVE AND GET -- FOR CODING IN THE DNA. IT'S A HIJACK OF AN ENZYME WHICH IS INITIALLY RECOMBINASE AND THAT'S MOSTLY DUE IN HUMAN VERT -- VERTEBRA CELLS. I DIVIDE THESE AND WE GO AS FAR AS WE CAN. IF I DON'T GO ALL THE WAY YOU'LL HAVE THE SLIDES TO LOOK AT AND I WILL BE HAPPY TO TALK TO YOU ABOUT IT. SO WE ESTABLISHED TOP 1. SO TOP 1 WAS DISCOVERED MANY YEARS AGO IN THE 70'S BY -- ONE OF HIS CONTRIBUTIONS WAS TO DISCOVER -- CALLED THE DNA UNTWISTING ENZYME. IT'S CALLED THAT WAY BECAUSE WHEN DNA WAS DETECTABLE WITH PROMADE LONG -- BROMIDE LONG AGO IN THE 70'S -- THE DNA'S NOT PURE, IT IS SUPER TWISTED. THEN IT BECAME FAIRLY STRAIGHTFORWARD TO DETECT THAT WITH THE BROMIDE. JIM -- DISCOVERED AN ACTIVITY IN THE MURINE EXTRACT IN SECONDS -- AND THAT'S WHAT TOP 1 DOES. SO TOP 1 WILL BIND THE SUPER CORD AND ATTACHED TO ONE END FREEING THE OTHER END AND ENABLING THE OTHER END TO JUST ROTATE AROUND THE INTACT BACKBONE. WHEN IS DNA FULLY REACTS, THE TWO ENDS ARE REALIGNED AND THEN THE TOP GETS AWAY. THIS REACTION OPERATES AT A SPEED IN RMP AT ABOUT 5 TO 6,000 RPM. IT'S AN EXTREMELY FAST ROTATION MACHINE. IT'S CONTROLLED WITH -- HEAR IN THE BUILDING WITH JUST A PAPER COMING OUT SHOWING HOW THIS IS ACTUALLY CONTROLLED. IT'S VERY FAST. IT DOESN'T TAKE ENERGIES. THERE'S NO RECURRENT -- THERE'S NO RECURRENT FOR METAL. IT WORKS AT ZERO DEGREES AND AS YOU WOULD EXPECT IT IS ESSENTIAL. IF YOU KNOCK OUT TOP 1 THE CELLS DIE IN THE VERTEBRATE, EXCEPT IN -- WHERE IT'S COMPLEMENTED BY OTHER -- IN THE VERTEBRA. SO THE WAY IT WORKS BECAUSE DNA -- AND YOU KNOW YOURSELF IF YOU TRY TO SEPARATE THE TWO STRANDS AND IF YOU ARE HOLDING ONE END, I WILL BE ABLE TO SEPARATE THEM TO A POINT. AFTER A WHILE YOU GET THIS MASSIVE AND I WON'T BE ABLE TO GO ANY FURTHER. SO WITH TOPOISOMERASE YOU WILL GET TRANSCRIPTION REPLICATION MACHINERY TO GET STUCK. SOME DAYS YOU HAVE COUPLING OF THE TOPOISOMERASE WITH THESE TRACKING PROCESSES THAT YOU COULD MOVE ON. AND THEREFORE YOU WILL RELAX THE DNA SUPER CORD ENABLING DNA MECHANISM AND TRANSCRIPTION. YOU WILL NOT GO VERY FAR AT ALL. SO TOP 1 RELAXES THE SUPER CORING WHICH IS BUILT UPSTREAM OR AHEAD OR REMOVING MACHINE AND IT WILL ALSO REMOVE THE NEGATIVE SUPER COATING WHICH IS GENERATED BEHIND. BOTH ACTIVITIES ARE ESSENTIAL. TOP 1 IS BOTH UPSTREAM AND DOWN STREAM FROM THE MOVING TRANSCRIPTION REPLICATION MACHINERY. SO TOP 1 BIEMEDZ THE SUPER COIL BY MAKING THIS SNAKE AND HOLDING ONE END AND LET THE OTHER ONE SWIVEL AROUND AND FULLY RELAXES THE SUPER CORED, RELIGATE. -- THIS COMPLEX IS INVISIBLE. IT'S SO FAST THAT IT'S SO TRANSIENT MANY SECONDS. BUT IF IT GETS SECOND IT GETS STUCK IN THREE CONDITIONS WITH DRUGS. THAT'S WHERE I WILL SHOW YOU THE ANTI-CANCER DRUGS WORK BY BLOCKING THIS. STALLING THIS. WHEN THE DNA ITSELF IS ALTERED AND THAT'S WHERE I WAS MENTIONING THE CARCINO GENEITY -- SO IN ADDITION TO DRUGS THE COMPLEXES CAN BE USED BY -- DNA LESIONS INCLUDING THE BASIC CARCINOGENIC AND VERY EFFECTIVE. SO THIS IS A DANGEROUS ACTIVITY FOR THE CELL. THERE ARE TWO GENES FOR TOP 1 IN HUMAN CELLS. THE FIRST GENE WAS KNOWN BY -- CLONING AT JOHNS HOPKINS BY -- GROUP WAS CALLED TOP 1. AND THE TYROSINE IS THE TERMINUS OF THE PROTEIN. THEN WE DISCOVERED ABOUT 12 YEARS AGO THE SECOND AND THE LAST -- GENE IN HUMAN CELL WHICH WE CALLED TOP MT BECAUSE THAT GENE THAT'S IN THE NUCLEUS SERVES ONLY THE GENOME IN ANY CELL. THERE ARE TWO GENOMES. THERE'S THE NUCLEAR GENOME WHICH IS MOSTLY 99% OF YOUR DNA. AND YOUR MITOCHONDRIA GENOME WHICH IS YOUR MOTHER'S INHERITED GENOME WHICH IS DEPENDING ON THE CELLS 1-5% OF YOUR GENOME. AND IT NEEDS TO BE DEALT WITH AND THIS TOP -- SOME VIRUSES AS TOP 1 AS WELL AND VERY FEW BACTERIA HAVE ALREADY DESCRIBED TO YOU THE MECHANISM OF SUPER CODING RELAXATION WHICH IS AN ESSENTIAL ACTIVITY OF THIS ENZYME, THAT'S WHY IT'S SO ESSENTIAL. AND THEN THE WHOLE FIELD CHANGED IN THE 1980'S WHEN IT WAS DIFFICULT COVERED THAT PREVIOUSLY KNOWN ANTI-CANCER AGENT CALLED -- FROM A CHINESE TREE -- WHEN IT WAS DISCOVERED THAT THIS DRUG -- IS EXQUISITELY TARGETED TO TOPOISOMERASE 1DNA COMPLEXES. WHEN THIS WAS DISCOVERED IN THE 80'S, THE DRUG COMPANIES IMMEDIATELY MADE -- AND IN THE UNITED STATES SMITH KLINE GENERATED -- AND IN JAPAN THE JAPANESE MADE A WATER SOLUBLE PRO DRUG WHICH IS A PRO DRUG WHICH IS CALLED -- WHOSE ACTION METABOLIZE -- I'M SORRY THIS IS WHAT IT'S CALLED BUT THIS REACTION METABOLIZE. THIS ONE ACTED BY ITSELF DOESN'T MEAN TO BE ACTIVATED BUT THIS ONE NEEDS TO BE ACTIVATED. AND IN KOREA THERE IS ANOTHER -- WHICH IS ALSO USED IN CLINICAL PROOF. THIS HAS BEEN APPROVED IN YEARS 2000 SO ABOUT 10 YEARS AGO OF RELATIVELY QUICKLY BECAUSE THE ACTIVITY WAS QUITE SPECTACULAR. TOP IS CURRENTLY USED IN THE UNITED STATES FOR PROOF OF OWE VARIANCE CANCER AND -- IS APPROVED FOR COLON CANCER. AND THERE ARE OTHER INDICATIONS AS CANCER AND PEDIATRIC CANCERS WHERE IT'S FAIRLY ACTIVE. THE WAY THESE DRUGS WORK IS QUITE REMARKABLE. THIS IS THE STRUCTURE OF CANCER -- SO IT LOOKS LIKE A PLAINER STRUCTURE WITH THE SIZE OF ABOUT -- WHICH IS FUSED IN THE MIDDLE. FOR A LONG TIME IT TOOK ANYONE TO KNOW HOW THESE DRUGS WERE IN TOP 1. WE KNEW THE ACTIVE ISOMER WAS TO PLANT NATURAL PRODUCT IS ONLY ONE WITH PRODUCTIVITY. THE REASON THIS DRUG WAS BLOCKING -- THIS IS SO TRANSIENT BUT IT JUST MOVES ALONG. BUT THIS BINDS AT THE INTERFACE, ONCE THE DNA'S OPEN IT CREATES A LITTLE WEDGE AND THE DRUG SHE'S VERY PLAINER CAN FIT RIGHT INTO THAT WEDGE AND SLOW DOWN. THIS WAS ACTUALLY PROVEN TO BE TRUE SO ABOUT TEN YEARS AGO THE STRUCTURE OF TOP 1 -- AND YOU SEE THE DRUG BOUND INSIDE THE TOP 1DNA COMPLEX. WHEN YOU TAKE AWAY THE SURFACE, YOU COULD SEE THE TOP -- EXACTLY LIKE DRAWN HERE EXCEPT SORT OF BECAUSE THE BREAK HERE IS AT THE BOTTOM. SO THE DRUG IS EXACTLY TRACKING THE TOP 1 COMPLEX. SO IF YOU LOOK AT ANOTHER WAY YOU CAN SHOW IT THIS WAY WHERE YOU HAVE THE DRUG MOLECULE WHICH IS RIGHT INTO THE COMPLEX. SO IT'S STUCK EXEN THE -- COMPLEX. WHEN THIS WAS REALIZED ABOUT TEN YEARS AGO AND THE REASON I'M GOING TO TELL YOU THIS IS IN -- THERE ARE TWO DRUGS IN CLINICAL TRIAL THAT WE DEVELOPED THAT ARE NOT THE TOP 1 INHIBITOR. SO THE DISCOVERY ASPECT -- WERE DISCOVERED WAS TO JUSTIFY THE FACT THAT ONE SHOULD LOOK FOR OTHER TOP 1 INHIBITORS. AND WE DEVELOPED THESE DRUGS FOR WHICH WE ALSO HAVE THE STRUCTURES AND THE DRUG IS SHOWN HERE, THE DNA HERE IS IN YELLOW. YOU COULD PRETTY WELL SEE THE BROKEN BACKBONE OF THE DNA HERE. THIS IS THE -- IS THE TYROSINE OF THE TOP SUMMARY. AND THE DRUG -- STUCK, TRAPPED THE TOP 1 COMPLEX AND THESE THE WAY THESE DRUGS INTOXICATE THE CELL. THEY USE THE ENDOGENOUS TARGET, TOP 1 TO POISON THE DNA AND MAKE PROTEIN LINK TOPOISOMERASE LINK THE DNA STAND. SO WE HAVE THE STRUCTURE FOR MANY DRUGS. BUT WHAT'S INTERESTED FROM THE CRYSTAL STRUCTURE AND THE ANALYSIS IS THE DRUGS HAVE TWO MAIN FEATURES BY WHICH THEY TRAP TOP 1 COMPLEX. THE FIRST FEATURE IS THAT THEY DO THIS BY STACKING. SO THEY MAKE THE SANDWICH BETWEEN, IT'S A FIVE PARTY INTERACTION SO IF YOU SEE SIDE WAY YOU SEE THE BROKEN DNA AND YOU CAN SEE THE DRUG IS EXACTLY FITTING INTO THE CLEAVAGE SITE. WE TEND THE DRUG INTO THE POSITION IS THE -- STACKING -- PHOBIC INTERACTION. THERE'S AN ADDITION AL SET OF INTERACTION BY WHICH THE DRUG IS HIGHLY SELECTIVE BECAUSE IT MAKES A NETWORK OF HYDROGEN -- OF THE TOP 1 ITSELF. THERE ARE THREE HYDROGEN BONES THAT ARE HIGHLIGHTED HERE AND WE KNEW FROM LONG TIME NOW THAT IF YOU MUTATE ANY OF THE HYDROGEN BOND CORRESPONDING AMINO ACID THE TOP 1 BECOMES VERY RETICENT TO CANCER PATIENTS. IN FACT, THAT TURNS TO BE TOTALLY TRUE BECAUSE THE PARADOX IS THE PLANT THAT PRODUCE -- AT TOP 1 AND THE PURIFIED TOP 1 FROM THOSE PLANTS -- FOR A WHILE IT WAS HARD TO UNDERSTAND HOW THESE PLANTS WOULD NOT BE KILLED BY THE TOXINS THAT WE'RE MAKING. SO INITIALLY EVERYBODY THOUGHT IT'S BECAUSE -- ONLY ACTIVATED IN THE BARK OUTSIDE OF THE PLANT TOP 1 IS. IN FACT WE KNEW IT'S NOT QUITE TRUE YOU GET A LOT OF -- RELEASE AND IN THE FLOWERS. AND WHAT TURNED OUT NOW ABOUT FOUR YEARS AGO IS IN JAPAN, THE GROUP DECIDED TO SEQUENCE ALL THE PLANTS THAT PRODUCE -- AND ALL THE PLANTS THAT PRODUCE IT AS ONE MUTATION IN THE TOP 1 WHICH IS -- AND IT WAS VERY STRIKING WHEN THIS CAME OUT BECAUSE YEARS BEFORE WE HAVE SELECTED A HUMAN LEUKEMIA CELL LINE FOR CANCER RESISTENT AND WE HAD PUBLISHED THE SELECTION OF THIS MUTATION THAT WENT INTO THE CELL LINE IMMUNE TO -- AND RENDERS THE PLANT IMMUNE TO THE TOXIN. THAT ALSO DEMONSTRATED CANCER -- TOTALLY SELECTIVE TARGETED BECAUSE IF YOU DO ONE MUTATION IN THE WHOLE ORGANISM IN THE PLANT, THE PLANT IS IMMUNE TO THE DRUG. IF YOU MUTATE TOP 1 IN CANCER CELLS, IT'S TOTALLY IMMUNE TO CANCER -- ONE MUTATION, ONE IMMUNIZED -- SO WE SET OUT SOME YEARS AGO THE DISCOVERY OF NEW TOP 1 INHIBITORS AND OUR ARGUMENTS TO DO SO IS WELL THE FIRST ONE IS THE CANCER -- SO MAYBE YOU SHOULD DO SOMETHING MORE. AND ALSO BECAUSE IT WAS KNOWN TO BE EFFECTIVE. IT DIDN'T TAKE A BRAINSTORMING EXERCISE TO THINK THIS WAS A LEGITIMATE TARGET. OR TWO, BECAUSE WE KNOW THAT DRUGS THAT TARGET THE SAME MOLECULE AT THE MOLECULE LEVEL COULD HAVE VERY DIFFERENT CLINICAL ACTIVITY. THE BEST EXAMPLE CLINICALLY IS THE -- INHIBITOR. IF YOU TAKE -- THEY BOTH TARGET THE TUBULIN -- THEY BOTH TARGET TUBULIN BUT -- IS A DRUG USED FOR GOUT. AND THIS IS USED FOR LEUKEMIA AND YOU WOULD NOT DO THE REVERSE. THE TARGET IS THE SAME BUT THE USE IS DIFFERENT. THEREFORE IT'S LOGICAL TO DEVELOP NON-CANCER -- BUT THE MAIN REASON OF ALL THE BASIC PHARMACOLOGY STUDY OF CANCER -- FIRST OF ALL -- THOSE LIMITING TOXICITIES. IN MICE, YOU CAN CURE MICE WITH -- THIS WAS PUBLISHED IN SCIENCE AT THE BEGINNING, OKAY. BUT YOU CANNOT CURE PEOPLE AND THE REASON YOU CAN NEVER GIVE ENOUGH DRUG BECAUSE YOU HAVE BONE MARROW AND THAT'S NOT TALK SEE. SO IT HAS ITS OWN LIMITED PROBLEM AND THEY ARE CHEMICALLY UNSTABLE. SO THE STRUCTURE OF -- IS INDICATED AS THIS PART HERE WHICH IS AS CONTAINED -- THIS IS VERY UNSTABLE. THIS IS OPEN AND GENERATES THE -- AND THE ONLY ACTIVE FORM -- I THINK THEY OVERSEE THE FACT THAT IT SLIDES TO 10% OF THE DRUG WHICH IS ADMINISTERED IS ACTUALLY TARGETING TOP 1. ALL THE REST IS THROWN OUT -- ELIMINATED WITHOUT ACTIVITY. IT'S HARD TO KNOW HOW MUCH REALLY. SO THIS IS A NON-RELIABLE TOXIN BUT CLINICALLY IT'S NOT VERY GOOD. SO THIS WAS A MAJOR LIABILITY. AND THE OTHER THING IS THAT THE CANCER -- COMPLEXES AND THAT'S WHY IN THE CLINIC THEY NEED TO BE ADMINISTERED THE INFUSION AND THEY ARE NOT ORALLY AVAILABLE. SO THEY ARE NOT VERY EASY TO USE IN THESE PATIENTS. SO WE SET OUT TO DEVELOPER THE NON-CAMPO -- IT'S VERY PROMISCUOUS SO THIS WAS TRIED IN PHASE TWO AND WAS ABANDONED BECAUSE IT HAS A TARGET EFFECT. THERE'S THE TOP 1 IN THE CLINIC, THIS CLASS HERE. THIS IS IN PHASE ONE, COMPLETING PHASE ONE -- SO THE SECOND CLASS WAS DEVELOPED BY GENSIME. SO IN PHASE ONE IS SECOND ACTIVITY SO WE LOOK FORWARD TO THESE DRUGS -- AND DIFFERENT SPECTRUM OF CLINICAL ACTIVITY. SO THE DISCOVERY OF THE -- WE DISCOVERED ABOUT TEN YEARS AGO, A LITTLE OVER TEN YEARS NOW WITH MARK KUSHMAN IN INDIANA. THE WAY WE DISCOVERED THIS DRUG, IN 1997 AND PUBLISHED IN 1998, YOU MAY KNOW THAT THE NCI HAS A LARGE DRUG DATABASE, ON HE EVER HALF A MILLION COMPOUND. AND IN 1998, I WAS VERY CURIOUS TO FIND SOMETHING IN THE DATABASE THAT COULD LOOK LIKE -- AND THUMPLAL ACTIVITY ACROSS THE CELL LINE BUT WOULD HAVE A DIFFERENT CHEMICAL STRUCTURE. AND WITHIN A FEW DAYS WE FOUND THESE COMPOUNDS WHICH WAS CALLED NS314622 BECAUSE THAT'S THE WAY THE COMPOUNDS ARE. THIS IS A NATIONAL NUMBER. IT'S A SINGLE NUMBER FOR EACH COMPOUND INCLUDING DRUGS THAT ARE ALSO CHEMICAL. AND MARK KUSHMAN PUT THESE DRUGS IN THE NCI BECAUSE IT WAS A BY PRODUCT OF -- AND HE DIDN'T KNOW WHAT TO DO WITH IT. SO THE DAY I CAME BACK AND I CALLED MARK KUSHMAN I DIDN'T KNOW AT THE TIME I THINK THIS COULD BE A TOP 1 INHIBITOR. WE LAUNCHED A TEN YEAR DISCOVERY PROGRAM MADE OVER 500 COMPOUNDS AND CAME DOWN TO TWO COMPOUNDS THAT ARE NOW IN CLINICAL TRIAL HERE. SO THESE ARE THE TWO COMPOUNDS. NOW WHEN YOU GO INTO CLINICAL TRIALS TODAY, IT'S NOT A SINGLE ENTERPRISE BECAUSE ALL OF THE STEPS WERE DONE ACTUALLY AT THE NCI. SO THESE TWO COMPOUNDS WHICH ARE CALLED LMP776 AND LMP400 HOW DID THEY GET FROM MY BENCH, OUR LAB TO BUILDING 10. WELL FIRST OF ALL WE HAD TO MAKE SURE THEY DID WHAT WE THOUGHT SO THEY HAD TO BE POTENT AS TOP 1 INHIBITORS AND WE HAVE ALL THE ASSAYS OF RECOMBINANT SO YOU DON'T HAVE TO READ ALL THE DATA. IT'S POTENT AS -- DANGEROUS CLEAVAGE COMPLEXES. AND WHAT'S INTERESTING IS THE SITES OF WHICH THE CLEAVAGE COMPLEXES WILL TAKE PLACE IN A CERTIFIED DNA. THIS IS IN THE SITE. YOU CAN SEE THIS IS CAMPO -- BUT YOU CAN SEE THE SITES LOOK DIFFERENT BUT IT MEANS ARE THEY GOING TO BE TALKING THE GENOME IN DIFFERENT PLACE. AGAIN, LOOK AT THE DATA, LOOK AT THE TITLE. THE SECOND THING WE WANTED TO DO IS THEY HAD TO WORK IN SALES. THEY HAD TO MAKE CLEAVAGES IN SALES. AND THE CLEAVAGE COMPLEXES HAVE TO BE PERSISTENT, MUCH MORE PERSISTENT THAN CAMPO -- THE THIRD THING WE WANTED TO DO IS OVERCOME THE DRUG REFLUX AND WE HAVE RESISTANCE SET UP WHICH IS OVEREXPRESSING EITHER THE -- OR THE NDL1NBCB 16789 WE USE THE CELL LINE TO LOOK FOR OUR COMPOUNDS. SO FOR EXAMPLE ABCG2 IS A MAJOR RESISTANT FACTOR FOR -- WE GET VERY VERY SMALL RESISTANCE, AND FOR THIS COMPOUND NO RESISTANCE. SO THAT WAS GOOD. AND THEN WHAT YOU HAVE TO DO IS OBVIOUSLY YOU HAVE THE TUMOR ACTIVITY. SO THE ANTI-TUMOR ACTIVITY TODAY TO LAUNCH A CLINICAL TRIAL YOU JUST CANNOT LAUNCH A CLINICAL TRIAL WITHOUT A BIOMARKER. IT WILL NEVER HAPPEN. YOU CAN ADJUST MEASURE SURVIVAL, DEATH, IT'S NOT GOING TO BE GOOD ENOUGH. EVEN IN PHASE ONE YOU HAVE TO MAKE SURE YOU'RE TARGETING THE CELL. SO WE DECIDED TO LOOK AT THE GENOME TARGETING USING A MARKER ONLY CHROMATIN DAMAGE WHICH IS HISTONE MODIFICATION CALLED HISTONE -- 2X WHICH IS NOW WORLD KNOWN AND IT IS THE FIRST MODIFICATION THAT WAS, THAT SHOWED THE NUCLEAR -- INDUCED BY DNA DAMAGE AND THAT WAS DISCOVERED BY WILLIAM -- SO HE'S A COLLEAGUE IN THE LAB. AND THEN WE DECIDED WITH -- WHO IS THE DIRECTOR OF THE -- DIAGNOSIS AND THE CLINICAL DIRECTOR FOR THE NCI, WE DECIDED TO SET UP THE BIOMARKER ASSAY FOR THE -- FOR THE USED WE WOULD USE IT FOR TRIAL. IT'S EASILY SAID BUT FIRST YOU HAVE TO DO IT IN MICE SO I'M GOING TO SKIP THERE BUT WHEN YOU HAVE GAMET 2X -- FOR INSTANCE THIS IS IN A PATIENT IN HAIR FOLLICLE. SO IF YOU PUT UP YOUR HAIR AND THESE ARE NUCLEI SHOWN IN RED, YOU HAVE, AND YOU HAVE HISTONE GAMETE -- THERE ARE ALWAYS A FEW. BUT THEN WHEN YOU FAKE A PATIENT THAT'S BEEN TREATED WITH THE DNA TARGETED AGENT, YOU COULD SEE ALL THIS NOW COMING UP. SO IT'S VERY CLEAR. SO YOU COULD USE -- BEEN USED TODAY. BUT PATIENTS DON'T LIKE IT VERY MUCH AND THE OTHER THING TOO IS IT'S SOMETIME UNRELIABLE. YOU DON'T KNOW HOW MANY HAIRS YOU NEED TO PLUCK TO GET A GOOD HAIR. YOU HAVE TO GET A NURSE WHO KNOWS HOW TO DO IT AND PATIENTS AMAZINGLY ENOUGH THEY WOULD RATHER HAVE A LITTLE SKIP BIOPSY THAN PLUCKING HAIRS. THE OTHER WAY YOU COULD LOOK IS THE BIOPSIES AND -- SO WE DECIDED TO SET UP AN ASSAY IN BIOPSY IN THE TUMORS THEMSELVES INSTEAD OF USING THE HAIRS AS SURROGATE. BUT WE HAD TO SET IT UP IN MICE WHICH WE DID. SO WE HAD TO PROFILE THE GAMETE'S RESPONSE. WE HAVE TO USE A REFERENCE DRUG WHICH WAS APPROVED FOR THE AND THEN USE MICE. ONCE WE WERE DOING THIS, WE WOULD DO IT WITH THE -- QUINNLYNN. THIS IS AN EXAMPLE OF FOUR DIFFERENT MICE TUMORS. THIS IS THE MICE SO YOU CAN SEE OCCASIONALLY THE GREEN STUFF. BUT WHEN YOU TREAT WITH QUINNLIEN, YOU COULD SEE NOW THE TUMORS LINING UP. SO THAT WAS GOOD. AND HAVING DONE THIS, THEN WE HAVE TO DEVELOP, AND THIS IS DIFFICULT TO TRANSLATE IN A NUCLEAR STAINING. WE DEVELOPED A CLINICAL GRADE SYSTEM WHEREBY ALL THIS WOULD NOT BE DONE BY HUMAN BUT BY A MACHINE WHERE YOU COULD SCORE OBJECTIVELY THE TUMORS ONCE YOU GIVE THE MACHINE THIS TUMOR VERSUS THIS ONE, IT'S GOING TO COME OUT WITH A NUMBER. SO WE SET IT UP IN THE MURINE SYSTEM FIRST AND THIS IS THE SIGNALS THAT WE WOULD GET FOR EXAMPLE IF YOU DON'T TREAT, YOU DON'T GET SIGNAL. AND AS YOU TREAT WITH DRUG YOU GET MORE AND MORE SIGNAL. AND SO ON. UNDER THE SAME CONDITIONS, WE MADE SURE THAT THE DRUGS WERE ANTI-TUMORAL. SO THIS IS THE GROWTH OF THE TUMOR IN THE UNTREATED MOUSE. THIS IS THE GROWTH OF THE TUMOR IN THE -- TREATED MICE. I'LL TRY AND FIND IT. YES, THIS ONE. THIS ONE. AND UNDER THESE CONDITIONS OF TOP T CAN THE RED, THIS WAS SO POORLY RATED THAT THE MICE LOST 20% OF THEIR WEIGHT. THEY WERE BARELY VIABLE AND THAT'S AS GOOD AS YOU COULD GET WITH TOP T CAN. UNDER THESE CONDITIONS WITH QUINNLYNN UNDER CONDITIONS OF THE MICE WE GET A MUCH BETTER RESPONSE. AND THEN THE RESPONSE COULD BE LOOKED AT WITH GAMETE STAINING. THAT HELPED US TO DETERMINE THE TIME WHERE WE SHOULD DO THE SECOND BIOPSY. SO AFTER ONE HOUR DRUG INFUSION, WE MEASURED DIFFERENT DIE, DIFFERENT CONCENTRATION OF DOSE TO DETERMINE WHAT WOULD BE THE BEST TIME FOR THE PATIENTS TO HAVE THEIR BIOPSY. AND WE SETTLED ON FOUR TO SEVEN HOURS WHERE YOU GET MANY GAMETE TO SIGNAL. SO AS OF TODAY, TWO -- QUINNLYNNS ARE IN CLINICAL TRIAL -- ACTUALLY BEING USED IN DRUG TRIALS. THESE ARE DRUG TRIALS WHERE CLINICAL TREATMENT OF DRUGS WITH LYMPHOMA AND HOW LONG. WE HAVE DEVELOPED POTENT SPECIFIC TOP 1 INHIBITORS. THEY ARE CLINICALLY STABLE WHICH TOTALLY OVERCOMES THE -- THEY DON'T NEED TO BE ACTIVATED. THEY INDUCE TOP COMPLEXES OF DIFFERENT SIZE THAN THE -- THE CLEAVAGE COMPLEX IS QUITE PERSISTENT. MUCH MORE THAN CAMPO -- WHICH ENABLES THE SINGLE ADMINISTRATION OF ONE HOUR HONEY FUSION. I WOULD HAVE LIKED TO MAKE ORAL DRUGS MAYBE THAT WILL BE THE NEXT SLIDE. THE DRUG RESISTENT ANTI-TUMOR, THEY ARE LESS TOXIC THAT IS CLEARLY SEEN IN THE MICE -- BEING USED AS A SENSITIVE BIOMARKER AND THE TRIAL IS ONGOING. SO THAT'S TOP 1. AND I'LL GO INTO THE TOP 2. SO IF YOU HAVE SOME QUESTIONS I CAN ANSWER NOW. >> [INDISCERNIBLE] >> THAT MEANS THAT TOP 1 HAS THE GENOME IN MANY PLACES AND IT HAS TO BECAUSE THE TRANSCRIPTION MACHINERY NEEDS TOP 1 ALWAYS UNRAVELING THE DNA IN FRONT AND BEHIND. SO IT CANNOT BE TOO LIMITED WHERE IT CAN WORK. BUT THE CAMPO -- POISONS THE COMPLEXES AT SPECIFIC PLACE. NOT ALL OF THEM. IT HAS TO HAVE A PARTICULAR BASE SEQUENCE. SO CAMPO -- ARE NOT PRESENT UNDERSTANDING. THEN THE QUINNLYNN POISON MOST SITES SO IT JUST POISONS THAT POSITION. WHETHER IT'S RELEVANT CLINICALLY WE SEE LATER BUT IT'S A GENOMIC TARGETING, IF YOU WISH. READY TO GO TO TOP 2? THAT'S MUCH HARDER. BUT IT WILL BE SHORTER. SO TOP 2, I GO BACK TO WHERE WE WERE. SO NOW I THINK YOU SHOULD BE STANDING HERE WITH THIS WAY OF DEALING WITH DNA SUPER COATING BY MAKING -- BREAKING ONE STRAND REVERSIBLY AND THEN THE CAMPO -- POISONING THE CLEAVAGE -- STILL WORKS AT ZERO DEGREES AND IS TRAPPED BY CAMPO AND -- QUINNLYNN. THAT'S THAT. WE MOVE TO THE TOP 2 SIDE. TOP 2 DIMER DOUBLE STRAND BREAK. SO TWO REASONS TO BE CALLED 2. HUMAN TWO GENES. AL KNOW AND BETA. BACTERIA -- TOP 4 TWO GENES. THE WAY IT'S GOING TO WORK IS MAKE A DOUBLE STRAND BREAK BY USING ONE SUBUNIT FOR EACH STRANLTD. THE WHOLE THING IS HOLDING AS A DIMER BUT EACH ONE WILL BE ONE STRAND IN CONVERTED MANNER. LINKAGE OF THE -- AND THEN WE SHOW YOU THE TOP 2 INHIBITORS AT THE TOP SITE -- SERVICING JUST MENTIONED BY WYNDHAM WILL DO WHAT CAMPO -- DOES FOR TOP 1 AND DOES IT FOR TOP 2 THEY'RE GOING TO BIND AS A DNA BREAK AND THEREFORE POISONS IT. THE DIFFERENCE IS TOP 2 BURNS ATP, NEEDS ATP. SO IT TAKES ENERGY. NEEDS MAGNESIUM. DOESN'T WORK AT ZERO. AND IT'S TRAPPED BY DIFFERENT DRUGS. THE DRUGS DO NOT OVERLAP. THE TOP 1 INHIBITORS DO NOT POISON TOP 2. THE TOP 2 INHIBITORS DO NOT POISON TOP 1. YET THEY HAVE THE SAME GEOMETRY IF YOU WISH BUT THEY DON'T GROW ACROSS. OKAY. SO IN THE TOP 2, WHY IS THERE TWO TOP 2'S? AL KNOW AND BETA. SO THE SECOND GENE OF TOP 2 WAS DISCOVERED AFTER THE FIRST ONE, AND WHAT IS THOUGHT TO BE NICE, TOP 2 ALPHA IS A REPLICATE ENZYME -- TOP 2 ON THE OTHER HAND IS A HOUSEKEEPING ENZYME THAT PROBABLY WORKS FOR TRANSCRIPTION. SO BETA IS MORE THE HOUSEKEEPING ENZYME 2 ALPHA IS MORE OF THE PRODUCT -- HOW DO THEY COMPARE? THEY ARE VERY SIMILAR. THIS IS THE SIMILARITY PERCENT BETWEEN TOP 2 ALPHA AND BETA. THE -- OF THE ENZYME SO THE ACTIVE SITES WHERE THE TYROSINE RESIDES FROM HERE, 93% SIMILARITY, VERY VERY CONCERNED. THE MAIN DIFFERENCE BETWEEN THE TWO ENZYME ON THE C TERMINUS AND IN THE N TERMINUS. AND THAT'S INTERACTING DOMAINS WITH OTHER PROTEINS AND THAT'S WHAT MAKES TOP 2 ALPHA -- A PROTEIN AND BETA A TRANSCRIPTION ASSOCIATED MACHINE. IN BACTERIA, THE CONSTRUCTION IS THE SAME EXCEPT THERE ARE TWO GENES TO MAKE ONE FUNCTIONAL PROTEIN MACHINE. IN THE CASE OF GYRATE -- IN THE CASE OF TOP 4 THERE'S PART E AND PART C. IT'S COMPLICATED IN MY PICTURE. BUT THEN YOU CAN SEE HOW SIMILAR THEY ARE IF YOU JUST THINK THEY WILL BE BUILT TOGETHER. IN BACTERIA YOU DON'T HAVE A DIMER YOU HAVE A -- THAT WILL BE THE SAME CONSTRUCTION. THESE THINGS ARE HIGHLY CONSERVED AND THEY USE MAGNESIUM TO JOIN THE DNA TO THE PROTEIN. YOU DON'T HAVE TO KNOW ALL THE DETAILS. SO HOW DOES TOP 2 FUNCTION? IF YOU THINK OF IT TO MAKE NOW WHAT IT'S GOING TO DO, IT'S NOT GOING TO LEAVE ONE STRAND IT'S GOING TO REALLY GO FOR IT. IT'S GOING TO CUT IT, HOLD IT, MAKE IT GO THROUGH AND AT THE END IT WILL BE THAT WAY. HOW DO YOU DO THIS? WELL THE WAY TOP 2 DOES IT, IT USES TWO GATES. IF YOU IMAGINE YOU HAVE TWO -- ONE WE CALL G FOR GATE AND THE OTHER ONE T FOR TRANSPORT. THIS IS THE STRAND THAT'S GOING TO GO ACROSS, OKAY. SO IF YOU IMAGINE I WANT TO GO THROUGH THIS. WHAT I'LL HAVE TO DO, I COULD DO THIS NOW BECAUSE IT'S BROKEN. THE ONLY WAY I COULD DO IT, YOU HAVE TO BREAK. IT'S GOING TO TAKE THIS SO THIS IS THE TRANSPORT STRAND AND THIS IS THE BREAK STRAND. I'M GOING TO TAKE IT THIS WAY. IN THE MEANTIME YOU HAVE TO OPEN BUT HOLD EVERYTHING. CAN'T LET IT LOOSE. SO THE ENTRY GATE AND EXIT GATE. SO THE ENTRY GATE IS HERE. IT WILL FIRST TAKE THE TWO STRANDS, BIND ONE, PUT ONE ON THE TOP, CURL AROUND IT AND THEN BREAK THE GATE STRAND IN THE PRESENCE OF MAGNESIUM. THEN THE OTHER STRAND GOES THROUGH. THEN IT GOES THROUGH. ONCE IT GOES THROUGH IT WILL RELIGATE THIS STRAND AND THEN OPEN ON THE OTHER SIDE AND FREE THE TWO STRANDS. SO NOW YOU HAVE DONE THE TRICK I SAID. TO DO THIS YOU BURN ATP AND YOU BIND MAGNESIUM. THE POWER OF THIS REACTION IS INCREDIBLE BECAUSE WHAT YOU COULD DO IS YOU COULD DECATENATE ANYTHING OR YOU COULD CATENATE OR YOU COULD, IF THERE'S A KNOT IN THE DNA, YOU COULD UNKNOT AND YOU COULD KNOT. YOU COULD ALSO RELAX THE SUPER -- BECAUSE IF YOU BREAK TWO STRANDS HERE YOU CAN RELAX EASILY. YOU CAN DO WHAT TOP 1 DOES. WHAT I'VE SHOWN HERE IS WHAT IS UNIQUE TO TOP 2, WHAT IS COMMON TO TOP 2 AND TOP 1, AND YOU COULD SEE THE COMMONALITY'S ONLY THE REACTION OF THE SUPER COATING. TOP 1 WILL NOT DO THAT. AND THEN GYRATING BACK HERE IS ONE MORE PROPERTY. IT COULD PUMP NEGATIVE SUPER COATING INTO THE GENOME. SO THE BACTERIAL GENOME ISSUE THAT OCCUR IS DONE BY GYRATE. WHY IS THIS IMPORTANT TO CLINICIANS AND TO YOU TO SOME EXTENT IS THAT TOP 2 INHIBITORS ARE COMMONLY USED BOTH ARE ANTI-CANCER AGENTS AND ANTI-BACTERIAL. SO IN THE ANTI-CANCER WORLD, DRUGS -- WERE USED WELL BEFORE. I CAME IN THE 80'S TO THE NIH HERE, WAS USING -- WAS A RED DRUG AND WE HAD -- WHICH WAS THE YELLOW DRUG AND THEN WE HAVE THE -- WHICH IS NO COLOR. BUT WE DIDN'T KNOW VERY MUCH, WE JUST KNEW THE NAMES. AND THEN LITERALITY TOP 2 WAS THE TARGET BECAUSE THE DRUGS WERE WELL BEFORE THAT. SO TODAY WHAT IS USED AS TOP 2 INHIBITOR, A TOP SITE VERY BROADLY USED -- MOST BROUGHTLY USED -- FOR BREAST CANCER AND THE OTHERS NOT THAT MUCH. THAT'S A LOT FOR CANCER -- THIS IS HUGE. SEVERAL YEARS AGO WHEN THERE WAS THE ANTHRAX REALLY SCARE, EVERYBODY WAS GOING TO THE PHARMACY GETTING -- OX LYNNIC ACID -- THIS IS A TREMENDOUS DRUG AND THEY ARE USED TODAY TO TREAT DRUG RESISTENT INFECTION INCLUDING TUBERCULOSIS. SO THESE DRUGS ARE VERY IMPORTANT IN THE CLINICAL MARKET WHICH IS THE MORE RECENT GENERAL -- FOR A WHILE WE JUST ASSUMED HOW THEY WORKED. AND ES ES -- IT'S ONLY THE LAST TWO YEARS THAT TOP 2 CAME TO LIGHT. IT CAME TO LIGHT EXACTLY AS EXPECTED. THIS IS THE CRYSTAL STRUCTURE OF THE HOMO DIMER OF TOP 2. TOP 2 LOOKS THE SAME SO THIS IS THE TOP OF THE MOLECULE WHERE THE STRAND WILL GO THROUGH THE FIRST GATE IN THE HOLE WHERE THE DNA WILL GO AND COME OUT THIS WAY. THE -- DNA YOU CANNOT SEE VERY WELL BECAUSE IT'S BURIED IN THE ENZYME BUT IF YOU TURN IT 90 DEGREES THIS WAY YOU CAN BETTER SEE THE DNA WHICH IS UP HERE. AND YOU CAN SEE EACH OF THE MONOMER. AND THE WAY THE DRUG BIND YOU CAN SEE THEM HERE. THERE ARE TWO DRUG MOLECULE, ONE IN EACH CLEAVAGE SITE AND THEY BIND. THIS IS THE DRUG HERE SO IT'S A TOP SITE WHICH IS A BASIS ON EACH SITE OF THE BREAK AS THE DNA OPENS BY THE ENZYME THE DRUG FALLS IN. IT'S NOT COVALENT AND TAKES HIGH CONCENTRATION WHERE IT WORKS AND BLOCKS THE ENZYME. AND AT ABOUT THE SAME TIME THE -- STRUCTURE OF THE BACTERIAL WHICH IS TOP 4 WITH THE QUINNLYNN THE FLOCKS SEEN WAS RESEAL AND THIS IS THE -- BUT YOU SEE IT LOOKS VERY MUCH THE SAME. THIS IS THE HUMAN TOP 2 BETA. THIS IS THE BACTERIAL PART TOP 4. THEY LOOK VERY MUCH ALIKE. EXCEPT THE BACTERIAL TOP 4 IS MADE OF FOUR PARTS INSTEAD OF TWO. THEY COME OUT TOGETHER THE SAME WAY. THE DNA WHICH IS BROKEN IS AT THE TOP. YOU ROTATE, YOU CAN SEE HERE AND HERE IS YOUR QUINNLYNN EXACTLY BOUND IN THE SAME PRINCIPLE WITH INTERACTION -- NETWORK OF HYDROGEN BOND TO THE TOP SUMMARY AND THESE, IF YOU MUTATE THAT'S HOW THE BACTERIUM OF COURSE RESISTENT AND YOU TAKE THOSE RESIDUE. SO WHAT THAT LED TO IS THIS CONCEPT OF DRUG THAT POISON THIS MACHINE, AND IN TERM OF DRUG DESIGN, THIS IS I THINK QUITE INTERESTING BECAUSE IT REVEALED THE INTERFACIAL INHIBITORS. SO IN THIS REVIEW CAME OUT THIS YEAR, IF YOU'RE INTERESTED YOU WILL FIND MANY MORE EXAMPLES BESIDES THE TOPOISOMERASE AN EXAMPLE LIKE -- INTEGRATE INHIBITORS ALSO WORK IN THIS MECHANISM. SO IN THE LAST MAYBE THREE MINUTES, I'LL JUST GIVE YOU A FLAVOR OF WHAT'S COMING UP IS THAT THESE THINGS THAT ARE BEING GENERATED ARE ACTUALLY DEALT WITH IN THE CELLS. IN THE CASE OF TOPOISOMERASE ONE CLEAVAGE COMPLEX THE WAY THEY DIE -- THE CLEAVAGE COMPLEX IS REVERSIBLE. THE CLEAVAGE COMPLEX CREATES A COLLISION SITUATION SO THE TOP IS STUCK AND AS THE TOP IS STUCK, THE POLYMERASE R NA OR DNA POLYMERASE IS PUSHING BEHIND. THE TOP DOESN'T MOVE FAST ENOUGH IT WILL GENERATE A COLLISION. AND THE COLLISION GENERATE IS DOUBLE STRAND N WHEN YOU HAVE THE -- DNA WHICH IS COLLIDING NOW THE DRUG CAN MOVE AWAY. YOU CANNOT LIGATE BECAUSE IT'S PAIRED WITH THIS DNA. TRANSCRIPTION IS THE SAME PROBLEM. SO THAT GAVE RISE TO THE NOTION THAT THIS MUST BE REPAIRED IN CELLS BY SOMETHING VERY SPECIAL. AND THIS TURNED OUT TO BE SHOWN INITIALLY AT NIH HERE BY HOWARD NASH WHO DISCOVERED THE FIRST ENZYME THAT IS ABLE TO UNHOOK TOP OF THE DNA. THE UNHOOKING ENZYME HOWARD NASH CALLED IT TYROSINE DNA FOR -- TIRE SEAL BECAUSE IT CLEARS THE TYROSINE FROM THE DNA AND -- BECAUSE IT'S THE -- REACTION. THE TOP 1 CLEAVAGE COMPLEX REPAIR NOW WE KNOW IS USING TWO MAIN PATHWAY, ONE WHICH IS TDP1 DEPENDENT AND ANOTHER ONE WHICH USES A BUNCH OF ANDO NUCLEASE. WHAT'S RELEVANT FOR YOU AS A CLINICIAN IS TP1 BUN TO DO THE REPAIR IS HIGHLY DEPENDENT ON PART. TDP1 IS ACTIVATED AND BROUGHT TO THE DAMAGE BY PARP AND IF YOU BROKE PARP WITH THE INHIBITOR OR THE NEW OTHER INHIBITOR -- OR THE BNM COMPOUND, WHAT YOU DO IS YOU PRESENT IT FROM WORKING AND THE CELL WILL ONLY -- AND THE CANCER CELL TURN OUT TO BE DEFICIENT IN ANY OF THESE PATHWAY, THEY WILL BE MORE VULNERABLE THAN A NORMAL CELL BECAUSE A NORMAL CELL, IF YOU BLOCK THIS, IT WILL STILL USE THIS. BECAUSE CELLS HAVE DEVELOPED REALLY VERY EFFECTIVE WAY TO DEALING WITH TOP 1 COMPLEXES. THEY ARE A FACT OF LIFE. BUT A CANCER CELL ALSO ENTERS MUTATIONS HERE AND THAT LED TO THIS WHICH WAS PUBLISHED LAST YEAR IS THAT, I DON'T WANT TO GO THROUGH THE COMPLICATION HERE BUT IT'S THE FACT THAT -- POLYMERASE COFACTOR OF TDP1 AND THEREFORE IF YOU BROKE PARP THE CELLS WILL TO NEED THE NUCLEASE AND THE IMPLICATION IS THAT CELLS THAT ARE DEFICIENT IN ERCC1 WHICH IS THE CASE OF I THINK A NUMBER OF TUMORS INCLUDING LUNG CANCER, THEN THE COMBINATION OF TDP1 INHIBITORS OR PARP INHIBITORS WITH TOP 1 INHIBITORS SHOULD BE SELECTIVELY EFFECTIVE. SO OTHERWISE YOU JUST INCREASE TOXICITY OF TOP 1 INHIBITORS. BUT TOP 2, WHAT COMES OUT TWO YEARS AGO IS THE TDP2. SO THE TOP 1, TOP 2 CLEAVAGE COMPLEX IS THE SAME THING. NOW WE KNOW, AND THIS IS JUST COMING OUT, JUST CAME OUT AND THERE'S ANOTHER PAPER COMING OUT VERY SHORTLY ON THE CRYSTAL STRUCTURE OF TDP2. THERE'S MACHINERY THAT WILL RELEASE THE TOP 2 FROM THE END. SO LET'S SAY IF YOU HAVE THE TOP 2 CLEAVAGE COMPLEX LIKE THIS WHICH IS LET'S SAY POISONED BY ANTI-CANCER DRUGS OR MAYBE NATURAL FLAVOR OF FOOD SUBSTITUTES OR DNA DAMAGE OR LEAGUES TOP 2 GETS STUCK. AND TO BE REPAIRED, YOU WILL NEED TO REMOVE THAT BIG TOP 2 FROM THE END SO YOU'LL NEED FIRST THE NATURE -- TO SOME EXTENT AND THEN TDP2 COULD COME IN. AND TDP2 CAN REMOVE THE FIVE PRIME LINKAGE, REGENERATING A -- PHOSPHATE WHICH COULD THEN BE LIGATED RIGHT AWAY AND THEN IS CELLS COULD SURVIVE. THE NOTION NOW IS SOME CANCER CELL COULD BE SELECTIVELY TARGETED, IF YOU COULD BLOCK TDP2 OR MAYBE IF TDP2 FOR SOME REASON IS DEFICIENT IN SOME NORMAL PEOPLE, THE PEOPLE MAY BE SENSITIVE TO TOP 2 INHIBITORS BECAUSE THEY CAN'T REALLY REPAIR VERY WELL. SO THERE'S A WHOLE FIELD THAT IS DEVELOPING BEYOND THE TOPOISOMERASE COMPLEXES. I WOULD LIKE TO FINISH HERE. I WILL BE HAPPY IF YOU HAVE QUESTIONS TO ANSWER HERE OR LATER OR BY E-MAIL IF YOU WANT TO COME AND SEE ME IN BUILDING 37. REALLY THE PEOPLE WHO IS MOST IMPORTANT CONTRIBUTORS I HAVE TO MENTION IS MY CURRENT -- MARK KUSHMAN -- AS I MENTIONED IS THE DEPUTY DIRECTOR FOR CLINICAL RESEARCH -- AND WITHOUT HIM I THINK WE WOULD NEVER HAVE BEEN BRAVE ENOUGH TO DEVELOP THE -- TOP 1 INHIBITORS. AND -- WHO HELPED ME INITIALLY TO DISCOVER THE -- QUINNLYNN. SO I CAN TAKE YOUR QUESTIONS IF YOU WANT. [APPLAUSE] >> WHAT HAPPENS IF YOU GIVE TOP 1 INHIBITOR AND TOP 2 INHIBITOR? >> WELL IT'S COUNTERPRODUCTIVE. YOU THINK IT WOULD BE GOOD BUT [INDISCERNIBLE] THEN YOU CANNOT GET THIS -- COLLISION. IT'S A GOOD IDEA BUT IT DOESN'T WORK AND THEN WE FIGURED OUT WHY. YOU NEED TO REPLICATE ACTUALLY. >> WELL AT'S GREAT. THANK YOU VERY MUCH. >> SURE. ALL RIGHT, THANK YOU.