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TTaarrggeettiinngg LLeeuukkeemmiiaa:: FFrroomm BBeenncchh ttoo BBeeddssiiddee

By Margie Patlak

TTaarrggeettiinngg LLeeuukkeemmiiaa:: FFrroomm BBeenncchh ttoo BBeeddssiiddee

Breakthroughs in Bioscience 1

Acknowledgments TARGETING LEUKEMIA-FROM BENCH TO BEDSIDE

Author, Margie Patlak

Scientific Advisor, Alan S. Rabson, M.D., Acting Director,

National Cancer Institute, National Institutes of Health

Scientific Reviewer, Janet Davison Rowley, M.D., D.Sc.,

Professor, Department of Medicine and Department of

Molecular Genetics and Cell Biology, University of Chicago

BREAKTHROUGHS IN BIOSCIENCE COMMITTEE

Fred Naider, Ph.D., Chair, College of Staten Island, CUNY

David Brautigan, Ph.D., University of Virginia,

Charlottesville

John Grossman, M.D., Ph.D., MPH, George Washington

University

Tony Hugli, Ph.D., La Jolla Institute of Molecular Medicine

Richard Lynch, M.D., University of Iowa College of

Medicine

Margaret Saha, Ph.D., College of William and Mary

BREAKTHROUGHS IN BIOSCIENCE PRODUC-TION TEAM

Director, Heather I. Rieff, Ph.D. Science Policy Analyst,

FASEB Office of Public Affairs

Editorial Coordinator, Paulette W. Campbell, Science

Writer/Editor, FASEB Office of Public Affairs

COVER IMAGE: Panel A is a bone marrow

sample from a patient with acute

leukemia� The dense� immature leukemic

cells outnumber the normal white blood

cells� which are smaller� A leukemic cell in

the center of the field is in the process of

dividing� as reflected by the presence of

two sets of chromosomes seen in the

nucleus of the dividing cell� Panel B is the

bone marrow sample from a leukemia

patient in total remission following thera�

py� The cells present are all normal bone

marrow cells and they show the diversity

in cellular structure that reflects the mix�

ture of cell types present in normal bone

marrow� Image @ �� Richard G� Lynch�

M�D�� University of Iowa�

PANEL A

PANEL B


n the 1960’s, many doctorswere drafted into the militaryunit of the Public Health

Service. Some of these menended up working on the chil-dren’s leukemia ward of theNational Cancer Institute’sClinical Center. The men neverencountered the jungles of VietNam and the horrors of war. But each day, after passing thewell-kept grounds of the ClinicalCenter and entering the newlybuilt brick building, they encoun-tered a different sort of horren-dous scene.

The children’s leukemia wardwas filled with the chatter of little ones, some as young as twoyears old, who, clutching theirteddy bears, toy cars or dolls,couldn’t expect to live more thana matter of months. One childmade a particularly indelibleimpression on her doctor. “I really want to finish the fourthgrade,” she confided in him. “Do you think I can?”

Four decades later, nearly 80 percent of all children withleukemia survive the disease—twice the survival rate in 1970.Impressive survival gains havealso been made for adults withthis type of cancer. Surprisingly,

many of the important stridesmade in the diagnosis and treat-ment of leukemia did not hingeon the efforts of cancerresearchers who specificallyaimed to conquer the disease, butrather by a diverse bunch of curi-ous scientists from pathologists,hematologists and immunolo-gists, to nutritionists, chemistsand geneticists. Theseresearchers, who, by pursuing theanswers to such basic questionsas “why do liver extracts cureanemia?”, and “how can wedetect life on Mars?”, collectivelyhomed in on the moleculardefects that underlie leukemiaand ways to counter those flaws.

White bloodThe success story of leukemia isactually a medical detective storythat got its start in 1845, whenRudolph Virchow, a young Berlinpathologist, was puzzled by thestrange set of symptoms dis-played by one of his patients. The50-year-old cook had been admit-ted to the hospital complaining offatigue, frequent nosebleeds andswelling of the legs and abdomen.She died just four months later.

Following an autopsy, Virchowslipped a slide of the patient’s

blood under a microscope.Peering into the eyepiece, he wasamazed to discover that thepatient had a lack of red bloodcells and an excess of white orcolorless cells. Virchow coinedthe term “leukemia”, or “whiteblood,” to describe the patient’scondition. He, along with theScottish pathologist John HughesBennett, who independently hadmade similar discoveries at thesame time, began a quest tounderstand what exactly goeswrong in this unusual disease.

A clue that helped solve thatmystery came about ten yearslater when another pathologist,Ernst Neumann, was conductingan autopsy on a patient who diedof leukemia. He discovered thatthe bone marrow wasn’t its nor-mal red color, but rather hadbecome “dirty green-yellow.”This, among other findings, ledhim to conclude that somethingawry in the bone marrow wasresponsible for the abnormalblood of leukemia patients. Italso led him to discover that thebone marrow was an importantsite for blood cell formation.

But many of the mysteries ofblood formation and leukemiawere not tackled until the innova-

LEUKEMIATARGETING LEUKEMIA:FROM BENCH TO BEDSIDE “It is that bridge between the clinic and the laboratory that sustains the ability to control disease�”

—Emil FreireichLeukemia (��) �� Suppl� �� S

I


tive use of cell stains at the endof the 19th century. These stainsenabled researchers to make finerdistinctions between the cellsthey saw under a microscope.This laboratory research led tothe surprising discovery thatblood cells, like the animals thatproduce them, do not start outfully mature and able to conductall the tasks performed by oldercells. Instead “ancestor” cellsgive rise to two types of primitivewhite blood cells known asmyeloblasts and lymphoblasts.Over time, lymphoblasts developinto the T or B cells that areknown for fighting infections.Myeloblasts eventually developinto a number of cell typesincluding those that fight infec-tions, foster allergic reactions, orhelp the blood clot.

Leukemia occurs when a whiteblood cell, whose development isfrozen, continues to duplicateitself. The resulting progeny ofcells are all in the same stage ofdevelopment and bear the distinc-tive hallmarks of the type ofancestral white blood cell thatgave rise to them.

Based on this understanding, by1900 leukemia was no longerseen as a single disease. Instead itwas imagined akin to a tree withtwo main limbs that in turn havetwo primary branches, all ofwhich reflect from what type ofcell the leukemia originates. (SeeFigure 1). One limb, myelogenousleukemia, has as its hallmark inthe blood and bone marrow eithera predominance of immaturemyeloblasts (one branch termedacute myeloid leukemia) ormature myeloid cells (otherbranch termed chronic myeloid

leukemia). With the other limb,lymphocytic leukemia, the bloodand bone marrow is overpopulat-ed by either precursor B or Tcells (one branch called acutelymphocytic leukemia) or matureB or T cells (other branch calledchronic lymphocytic leukemia).

Despite their divergent origins,all types of leukemias share thesame set of symptoms, just asfever and congestion can be thesymptoms for a number of differ-ent microbial infections. When

people develop leukemia, theirabnormal white blood cells crowdout or hamper the functioning oftheir red blood cells, fostering thetiredness and paleness that are thehallmarks of anemia. These whiteblood cells also do not effectivelyfight infections, which can befatal. A lack of functioning cells(platelets) that clot blood makespeople with leukemia prone tolife-threatening bleeding

episodes. Leukemia cells thatcongregate at various spots in thebody can also spur a variety ofother symptoms, including boneor joint pain, or enlarged organs.Untreated acute leukemia pro-gresses rapidly to death, whereasuntreated chronic leukemia canbe longer lasting, although just asdeadly in the long run.

Although leukemia afflicts bothadults and children, by the 1920’sit was recognized that the diseasemostly attacks children. At about

this time, leukemia began analarming rise in incidence. In1930 it was considered a relative-ly rare disease. But by 1960, sta-tistics collected in Great Britainrevealed that leukemia hadbecome the second leading causeof death in children.

This rising incidence ofleukemia in children reflects theexplosion of knowledge aboutinfectious diseases that began

FFiigguurree �� LEUKEMIA TREE By �� leukemia was no longer seen as a single disease�but rather akin to a tree with two main limbs that in turn have two primary branches�all of which reflect from what type of blood cell the leukemia originates�


with the pioneering work ofRobert Koch and Louis Pasteur.At the end of the 19th century,these scientists started the searchfor the microbial causes of manycommon contagious diseases. Bythe middle of the 20th century,researchers had pinned down theculprits responsible for many ofthe infections that were lifethreatening to children, includingdiphtheria, pneumonia, and tuber-culosis. Their findings fosteredthe development of antibioticsand vaccines to cure or preventmany of these ills, allowingleukemia to come to the forefrontas a major cause of death amongchildren.

Leukemia’s emerging impor-tance, however, was not accompa-nied by an improved understand-ing of what caused the disease orhow it could be treated. Indeed,when leukemia first became morecommon, researchers were stilldebating whether it was an infec-tious disease or a cancer. Thislack of understanding ofleukemia hampered the develop-ment of effective drugs for thedisease, which quickly wreakedits tragic effects. In 1936, chil-dren lived only a few weeks afterbeing diagnosed with leukemia.

Dreaming of a cureBut hope was on the horizon,thanks to a Harvard doctor whowas curious as to what it was inliver extracts that cured hispatients of anemia that was notcaused by leukemia. He testedthe newly discovered vitamin,folic acid, which is abundant inliver, and found it fostered theproduction of red blood cells in

his patients. Following his lead, anumber of doctors started to usefolic acid to treat the anemia theirleukemia patients experienced.But Sydney Farber, a Bostonpathologist, noticed that patientsworsened when they were giventhe vitamin. This gave Farber theidea that if folic acid acceleratedthe progress of leukemia, maybethe disease could be slowed downby a compound that countersfolic acid in the body.

A persuasive man, Farber con-vinced chemists at LederleLaboratories to concoct a com-pound that would block theaction of folic acid. This was pos-sible because laboratoryresearchers had recently elucidat-ed the structure of the vitamin.Using this information, scientistsat a pharmaceutical company cre-ated an inactive folic acid mimicthat, by substituting for the realthing, prevented folic acid fromsubstantially affecting blood cells.

In 1947, despite the objectionsof many, who thought childrenwith leukemia should be left todie in peace, Farber and his col-leagues began treating a smallnumber of children with acutelymphocytic leukemia (ALL)with the folic acid mimic, calledaminopterin. The researcherswere encouraged when a majorityimproved, including one childwho showed no signs of the dis-ease for several months.Referring to this child, Farber’scolleague Robert Mercer reflect-ed in a recent issue of the scien-tific journal Medical PediatricOncology, “The bone marrowlooked so normal that one coulddream of a cure.”

Unfortunately, none of thepatients given aminopterin werecured of their leukemia. But thelimited success of theseresearchers sparked others to pur-sue a search for drugs that couldstymie the progress of leukemia.

Discovery of the next leukemiadrug stemmed from the laborato-ry findings that cells need DNAto divide, and that one of themajor building blocks of DNAare compounds called purines.Building on that information,chemists George Hitchings andGertrude Ellion of WellcomeResearch Laboratories concocteda purine look-alike called 6-mer-captopurine (6-MP). Like a mis-shaped object in a factory assem-bly line, this compound jammedDNA production and stoppedleukemia cells from dividing.

Other important early anti-leukemia drugs were discoveredwhen laboratory researchers werepursuing seemingly unrelatedavenues. For example, testing onrats the effects of a folk remedyfor diabetes—an extract of theperiwinkle plant—led to the dis-covery of vincristine. Otherresearch aimed at seeing ifextracts from adrenal glandscould mimic the beneficialeffects of pregnancy on rheuma-toid arthritis led to the discoveryof another drug called pred-nisone. These compounds aremainstays in many anti-cancerdrug regimes.

But none of these drugs, singly,were able to save children withacute leukemia despite their teas-ing ability to temporarily eraseany obvious signs of the disease.What made the leukemia come


back? This was not only anintriguing puzzle, but also a life-threatening dilemma to the chil-dren afflicted with the disease.

It took an innovative anddiverse team of clinicians, phar-macologists, and geneticists tomake any headway into solvingthat puzzle. One member of theteam, geneticist Lloyd Law of theNational Cancer Institute (NCI),developed a way to studyleukemia in mice that provedimmensely valuable in ascertain-ing why the remissions were sofleeting. Another team memberwas pharmacologist HowardSkipper, then at the Southern

Research Institute, who used tosay, “a model is a lie that helpsyou see the truth.”

Skipper applied pharmacologyprinciples and mathematicalmodeling techniques to findingsfrom Law’s leukemic mice toshow that although the new can-cer drugs might kill nearly allleukemia cells so that no residualdisease could be detected viastandard means, a small numberof leukemia cells was left lurkingbehind. These cancer cells couldeventually multiply to the pointof causing a resurgence of thedisease. This suggested that ifdoctors wanted to cure their

patients of leukemia, they had tocontinue to treat them even aftertheir symptoms disappeared.

Another key piece to the puzzlecame when Law discovered thatwhen he treated his leukemicmice with 6-MP, their leukemiacells eventually stopped respond-ing to the drug. But a glimmer ofhope came from the additionalfinding that although theseleukemia tumors developedresistance to 6-MP, they becamemore sensitive to the folic acidmimic methotrexate. This drugreadily killed the leukemia cells.

Law’s intriguing findings sug-gested to NCI hematologists and

Christian Neumann Sydney Farber Howard Skipper Emil J. Fredriech, Jr. Emil Frei III

TI

MELINE

1845-PathologistsRudolph L�K� Virchowand JohnHughesBennettfirstdescribeleukemia asa medicaldisorderwith distinc�tive hall�marks in theblood�

1850s-PathologistErnstChristianNeumanndiscoversthat thebone mar�row isabnormal inleukemiapatients�

1900-Innovativecell stainsreveal thatleukemia isnot a singledisease� buta collectionof at leastfour differ�ent types�

1940s-PathologistSydneyFarber dis�covers thatan inactivefolic acidmimic(aminopterin)can tran�sientlyimprovesome chil�dren withleukemia�

1950s and1960s-Basicresearchleads to thediscoveryof severalanti�leukemiadrugs�including �mercapto�purine� vin�cristine�and pred�nisone�

1950s and1960s-GeneticistLloyd Lawand phar�macologistHowardSkipper dis�cover whyleukemiarecurs whenpatients aretreated withjust onedrug�

1962-The firstleukemiacures areachievedwhen EmilJ� Freireich�Jr� and EmilFrei IIIbegin treat�ing patientswith combi�nationchemother�apy�

1960-PathologistPeterNowell andDavidHungerforddiscoverthat in thecancerouswhite bloodcells of sev�eral patientswith chronicmyeloidleukemia� aportion ofone of theirchromo�somes ismissing�

Rudolph L. K. Virchow

FFiigguurree ��


clinical researchers Emil J.Freireich Jr., and Emil Frei IIIthat they might get the bestresults if they treated patientswith all four anti-leukemia drugscurrently available: vincristine,amethopterin, 6-MP, and pred-nisone. They began treatingpatients with that regimen in1962. Many of these patients arestill alive today—living testimonyto Farber’s audacious decision totreat patients with leukemiainstead of merely allowing themto die.

However, another major stum-bling block remained. The major-ity of patients experiencing com-

plete and long-lasting remissionseventually developed an excess ofleukemia in their brains thatkilled them. Going back to thelab bench, researchers discoveredwhy—the drugs that were soeffective at killing leukemia cellsweren’t penetrating into thespinal cord or brain. Leukemiacells flourished in these centralnervous system hideouts, eventu-ally causing fatal complications.Fortunately, animal studiesshowed a way out of this predica-ment—direct injection of the can-cer drugs into the spinal-cordcanal and irradiation of the head(brain).

Additional drugs and improve-ments in supportive therapy alsohelped bring the cure rate ofchildhood ALL up to its presenthigh rates. But even when radia-tion and drug therapy cures chil-dren of leukemia, the treatment isso toxic that it can cause a num-ber of serious complications,including the development of anew type of leukemia or braintumors or heart problems. Part ofthe toxic nature of the therapyresides in the fact that it is notspecific—it doesn’t affect justleukemia cells, but any activelydividing cell. Not all types ofleukemia, in addition, are curedby the regimen that is usually

1970-The firstflowcytometer isdevelopedby immu�nologistLeonardHerzenbergand his col�leagues�

1973-HematologistJanetRowley discoversthat chronicmyeloidleukemiagets trig�gered by apiece ofchromosome�� breakingoff and reat�taching ontochromosome� in a whiteblood cell�

1980s-Researchersdiscover thefused gene(BCR�ABL)that is createdwhen a portion ofchromosome�� translo�cates to chro�mosome ��

1980s and1990s-The use offlow cytom�etry revealsmore than adozen differ�ent subtypesof leukemiaand aids thedetection ofleukemiacells surviv�ing treat�ment�

1985-BiochemistKary Mullisfirst con�ceives ofpolymerasechain reac�tion (PCR)�

1990s-PCR is usedto fine�tuneleukemiadiagnosisand detectleukemiacells thatsurvivetreatment�

1990s-OncologistBrian Drukerdiscovershow BCR�ABL triggersexcessivedivision ofwhite bloodcells andshows thatSTI�� acompounddevelopedby Novartis�has anti�BCR�ABLactivity incell cultures�

2001-Afterhuman test�ing revealsSTI��’seffective�ness� theFood andDrugAdmin�istrationapproves itunder thenameGleevec� asa drug forchronicmyeloidleukemiapatients�

Leonard Herzenberg Janet Rowley Kary MullisPeter Nowell Brian Druker


effective for childhood ALL. For example, the chemotherapyregimen for ALL is not usuallyeffective for chronic myeloidleukemia (CML).

Genetic cluesTo develop more targeted andnon-toxic treatments for varioustypes of leukemia, scientistsneeded to delve deeper into theconundrum of what exactly caus-es them. An important clue thatguided this journey was thatexposure to high amounts of radi-ation, such as that emitted by theatomic bomb dropped inHiroshima, damaged the gene-harboring chromosomes of sur-vivors and made them prone todeveloping leukemia. This sug-gested that the root of a cancercell’s wayward behavior lies inthe genetic machinery of the cell.

A University of Pennsylvaniapathologist Peter Nowell provideda view into that genetic machin-ery, in 1960. He was trying todiscern chromosomal abnormali-ties in leukemic white bloodcells. To rid blood specimens ofred blood cells so he could studythe remaining white blood cells,Nowell treated the specimenswith a plant compound calledphytohemaglutinin. This chemicalcaused the red blood cells toclump together for easy removal.

One day, when he went to col-lect the blood of a leukemiapatient for his research, he dis-covered the patient had gone intoremission. But when Nowellexamined the patient’s whiteblood cells he was surprised tosee evidence that they were divid-ing. At the time, it was thought

that normal white blood cells inthe blood were incapable ofdividing, unlike cancerous whiteblood cells. So Nowell decided tosee how these patients’ cells compared to the normal whiteblood cells found in his ownblood.

When Nowell peered under themicroscope to examine his whiteblood cells, he was surprised todiscover stretched across theirnuclei in an orderly fashion, 23pairs of worm-like structures heimmediately recognized as chro-mosomes. When a cell divides,its chromosomes must disentan-gle themselves from their usualincomprehensible nuclear jumbleand line up in the duplicate pairsthat Nowell saw in his own cells.By essentially catching them inthe act of reproducing, Nowellhad a detailed view of humanchromosome structures in normalblood cells.

After a process of elimination,Nowell discovered that the plantcompound he used to sift out hisred blood cells had stimulated hiswhite blood cells to divide. Thiscompound opened the door to theeasy study of how the chromo-somes of leukemic white bloodcells compare to those of normalwhite blood cells. In 1960,Nowell and his colleague DavidHungerford reported that in thecancerous white blood cells ofseveral patients with CML, a por-tion of one of their chromosomeswas missing.

Where had it gone?

This was a mystery until adozen years later when innovativestains fostered unique stripingpatterns that researchers could

use to identify each chromosome.Using these stains, cytogeneticistJanet Rowley of the University ofChicago noticed that in the whiteblood cells of patients with CML,chromosome 9 was longer thannormal and the additional lengthhad a striping pattern just likethat of the portion of chromo-some 22 that was missing inthese patients.

Rowley had found the smokinggun—the cutting and pasting of apiece of chromosome 22 ontochromosome 9 apparently trig-gered these patients’ leukemias.But why was this “translocation”a problem?

It took nearly two decades andthe basic research of several labo-ratories to answer that question.The winding DNA that comprisechromosomes have sections(genes) that direct the production

Figure �� An image of a dividing cell

from a patient with chronic myeloid

leukemia (CML)� CML is triggered upon

the cutting and pasting of a piece of

chromosome �� onto chromosome ��

resulting in a protein called BCR�ABL�

The above cell shows the BCR probe

labeled in green on the normal chromo�

some nine and the ABL probe labeled in

red on the normal chromosome �� The

abnormal chromosomes � and �� are

labeled with a fusion signal that is green

and red with a yellow area of overlap�

Image @ �� Janet D� Rowley�

University of Chicago�


of specific proteins. But much ofthe chromosomes are comprisedof barren stretches of DNA thatapparently lack genes. However,research revealed that when thetranslocation that Rowley detect-ed occurs, chromosome 22breaks off right in the middle of alength of DNA that is a gene. Totop it off, this chunk of chromo-some lands smack in the middleof another gene on chromosome9. The fused genes produce a newprotein called BCR-ABL. Thisprotein quickly rose to fame as amajor suspect in the cause of CML.

In the 1980’s, a research teamled by the molecular biologistDavid Baltimore, then at theWhitehead Institute forBiomedical Research inCambridge, set out to learn moreabout this suspect. His lab’sresearch indicated the BCR-ABLprotein was a kind of enzymecalled a tyrosine kinase.

This discovery set off bells inbiochemistry labs because labo-ratory research has revealed thattyrosine kinases play a key rolein governing the proper growthand division of cells. It is precise-ly these processes that go awry inleukemia and other cancers.Nearly 50 tyrosine kinases havebeen implicated as playing keyroles in the development of vari-ous types of human cancers,including leukemia, breast andbladder cancer. Consequently,tyrosine kinases have become hotnew targets for anticancer drugs.(See sidebar, Tyrosine Kinasesand Cancer, on page 9).

One researcher prompted topursue the tyrosine kinase trailwas Brian Druker, an oncologist

who became fed up with thestate-of-the-art of cancer care.About the time that Baltimorepegged BCR-ABL as a tyrosinekinase, Drucker decided to ven-ture into the lab at the Dana-Farber Cancer Institute in Bostonto gain a better understanding ofhow exactly BCR-ABL mighttrigger leukemia.

Laboratory research hasrevealed that tyrosine kinasesusually trigger cell growth ordivision by activating key mole-cules known as signaling pro-teins. The kinases prompt thesesignaling molecules into actionby decorating them with phos-phates that they remove from

ubiquitous molecules in cellscalled ATPs. This process isknown as phosphorylation.

What did BRC-ABL phospho-rylate? Druker recognized thatthe answer to this question wouldbe key to understanding how thefused genes cause leukemia. Sohe compared the phosphorylatedcompounds generated by bloodcells that had the BCR-ABLfusion gene inserted, to the phos-phorylated compounds made byblood cells that lacked the gene.

This led Drucker to discoverthat the BCR-ABL protein acti-vates a molecule (rasGTPase acti-vating protein) that plays a keyrole in prompting cells to divide.

Schematic ofCML white blood cells

FFiigguurree �� SCHEMATIC OF DEVELOPMENT OF CMLChronic myeloid leukemia is triggered when within a white blood cell a portion of chro�mosome �� breaks off and reattaches to chromosome �� This causes the BCR gene fromchromosome �� to attach to the ABL gene of chromosome �� The BCR�ABL fused genedirects the production of a protein that prompts the white blood cell and all its progenyto divide excessively� Designed by Corporate Press�


Unlike normal tyrosine kinases,however, BCR-ABL lacks a vitalsection that enables it to eventu-ally shut off and stop the chain of events that results in cell division. Like a stuck throttle,BCR-ABL revs up certain whiteblood cells, prompting them tocontinually divide. The end resultis CML.

With this information in hand, Druker teamed up withresearchers at Ciba-Geigy, now known as Novartis. Thesescientists had concocted several compounds designed to inhibitthe activity of various tyrosine kinases. They handed these compounds over to Druker, who tested their ability to inhibit theactivity of BCR-ABL in bloodcells that had the fused gene. One of these compounds, calledSTI-571, showed promise.

In 1998, Druker and his colleagues began testing STI-571on CML patients who weren’tresponding to the immune stimu-lant interferon, which was thegold standard of therapy for thistype of leukemia even though ithas many serious side effects andfails to cure the majority ofpatients. Three years later, theresearchers were amazed toreport that STI-571 had provedsafe and effective for an amazing53 of 54 patients who received itin high doses. Some patients,who were practically planningtheir funerals at the beginning ofthe study, felt remarkably betterafter taking the drug for just afew months. Equally impressiveis the fact that no serious sideeffects of STI-571 have beenreported yet.

Although there are more than a hundred different kinds of cancers� each with theirown unique hallmarks� all cancers can be characterized by an excessive amount ofcell proliferation� Indeed� cancer cells become deaf to the normal controls on cellgrowth and continually divide�

Insight into what causes that lack of control has come from laboratoryresearch on normal cell division and what steps in that process are corrupted in cancer cells� Compounds known as growth factors are manufactured in the body to stimulate cell division and cell growth� These compounds bind to antenna�likegrowth factor receptors lodged in cell membranes� When a growth factor attachesto its receptor� it triggers a cascade of chemical signals inside the cell that carry the message that cell division is needed to the cell nucleus� which starts the cellsplitting process� Key molecules in this signaling pathway are tyrosine kinases�

These enzymes often act as chemical messengers between growth factor receptors and other signaling proteins that stimulate cell division� To prompt thesesignaling molecules into action� tyrosine kinases transfer phosphate groups fromATP� a molecule that is abundant in all living cells� onto specific sites on the signal�ing molecules� These activated proteins in turn prompt cell growth and division�

The actions of tyrosine kinases in normal cells are closely controlled by growthfactors and other enzymes called tyrosine phosphatases� which remove the phos�phates and act as brakes to arrest growth and division� But in cancer cells� the tyro�sine kinases are often overproduced or made in forms that are active all the timeand no longer dependent on growth factors� Some cancer cells also lack functionaltyrosine phosphatases� Because they lack these brakes on cell growth and division�cancer cells continually divide resulting in tumors� or in the case of leukemia� exces�sive amounts of certain kinds of white blood cells in the bloodstream and bonemarrow� To counter that excessive cell division� many new chemotherapy com�pounds target the actions of tyrosine kinases�

For more information� refer to Jensen�Blume� P� & Hunter� T� �� Oncogenickinase signalling� Nature; ��:��

Tyrosine Kinases and Cancer


It’s too early to tell if STI-571,which goes by the trade nameGleevec, can improve the long-term survival of CML patients.But the remarkable results so farprompted the Food and DrugAdministration to approve it foruse in these patients.

Gleevec is just one of severalpromising treatments forleukemia that have recently beendiscovered because laboratoryresearch has uncovered the causeof a specific leukemia. For exam-ple, Rowley discovered that theleukemia trigger for patients withacute promyelocytic leukemia(APL) is a translocation betweentheir chromosomes fifteen andseventeen. This chromosomeswapping disrupts the normalfunctioning of a receptor forretinoic acid, a form of vitaminA. It is thought that because thereceptor does not work properly,the cells are essentially starvedfor retinoic acid. This compoundstimulates the maturation of cer-tain cells and, when it is given toAPL patients, it enables theirleukemia cells to develop nor-mally, thereby boosting long-termsurvival.

However, findings in somepatients suggest that the effec-tiveness of both retinoic acid andGleevec may be hampered by thedevelopment of drug resistance.Thus, these drugs may have to becombined with others to generatecures for these leukemias.

But many agree that this strate-gy for combating leukemia holdsgreat promise. In the two decadessince Rowley and her colleagueslearned that a specific chromoso-mal translocation gives rise to aspecific type of leukemia, she

and other researchers have pin-pointed over a hundred differenttranslocations in the cells of vari-ous leukemia patients. Scientistsare beginning to decipher whichof these translocations are vital incausing leukemia. The presenceof various common translocationsis already being used to classifypatients’ leukemias. This classifi-cation scheme has provenextremely valuable in decipheringwhy two patients, whoseleukemias fall under the samebroad category of leukemia (ALL,for example), can have such dif-ferent responses to treatment. Ithas also led to more tailored treat-ments, as some drugs work betteron leukemia cells with certainkinds of translocations.

The ability to make fine dis-tinctions in leukemia types is rev-olutionizing the treatment ofpatients with these cancers. Thisis possible not only because ofthe advances made in uncoveringthe genetic causes of leukemias,but also stems from the work ofresearchers from an unusual pot-pourri of disciplines fromimmunology to physics. Thesescientists weren’t bent on findinga cure for cancer, but rather had adiverse bunch of objectives,including developing equipmentto detect extraterrestrial life andtrying to find the genetic basisfor antibody diversity.

Go with the flowA device that has provenimmensely valuable in leukemiadiagnosis and treatment, the flowcytometer, was dreamed up byspace scientists who wanted anautomatic way to sift out extrater-restrial life from materials col-

lected from Mars. To meet thattask, the National Aeronauticsand Space Administration(NASA) funded the research of amultidisciplinary team of scien-tists at Stanford University. Butas support for the research teamshifted from NASA to NIH, theresearch emphasis shifted fromdesigning a device useful tospace exploration to developingone that would impact the med-ical arena—specifically a devicethat could help diagnose therejection of transplanted organsby the immune system.

By 1970, the researchers led byimmunologist LeonardHerzenberg debuted their proto-type flow cytometer. This devicehad the amazing ability to chan-nel a stream of a mixed popula-tion of cells so that the cells tum-bled single file past a laser light.The cells were sorted based onhow they scattered the laser lightand by how much of a fluores-cent dye they absorbed and emit-ted. The flow cytometer not onlyseparated the various cell typesfor further study, but also count-ed how many of each cell typewas in a sample. This devicecould sort cells at the remarkablerate of 5000 cells per second!

The researchers further refinedtheir cell sorter by attaching anti-bodies to a fluorescent dye. Theseantibodies selectively bound tospecific cell types, boosting theirdegree of fluorescence. But theresearchers had trouble standard-izing the original crude antibodymixtures they used because thesemixtures reacted to more thanone cell type and varied frombatch to batch. This lack of stan-dardization was limiting the use-fulness of the flow cytometer.


Fortunately, a pair of immunol-ogists trying to uncover thegenetic basis of antibody diversi-ty came to the rescue. For theirlaboratory research, CesarMilstein and Georges Kohler,working in Cambridge, England,needed antibody-forming cellsthat could survive and perpetuatethemselves for a long time in thelaboratory cultures used for theirexperiments. So they fused anti-body-forming cells with tumorcells, which are capable of main-taining themselves for long periodsoutside the body. The end resultwas a hybrid cell that not only waslong-lived in the laboratory, butalso produced only one type of (or“monoclonal”) antibody.

In 1976, Herzenberg spent sev-eral months in Milstein’s labora-tory and immediately realizedthat these novel monoclonal anti-bodies were the solution to theproblem he had standardizing hisflow cytometer. He began usingthe monoclonal antibodiesattached to fluorescent dyes inhis flow cytometer severalmonths later.

Researchers then usedHerzenberg’s refinements on flowcytometry to discern the varioustypes of T and B cells. The sur-face of each of these types, aswell as those of other white bloodcells are studded with a distinc-tive array of proteins known asantigens. These telltale landmarksare invisible in stained cellsviewed under the microscope.But they can be easily spottedwith a panel of different fluores-cent monoclonal antibodies. Eachof these antibodies is designed to

latch onto a specific cell-definingantigen and send out a red,orange or green glow that can bedetected by the flow cytometer.

Thanks to the powerful combi-nation of monoclonal antibodiesand flow cytometry, researcherswere able to refine the four basiccategories of leukemia into morethan a dozen different subtypes.These subtypes are classifiedaccording to the pattern of pro-teins they display on their cellsurfaces.

The various subtypes ofleukemias laboratory researchershave uncovered using flowcytometry do not merely satisfythe scientific instinct to order andclassify, but rather are revolution-izing the care of leukemiapatients. Studies reveal that effec-tive treatment of some of thesesubtypes require higher doses ormore extensive or prolongedchemotherapy regimens. Theleukemia cells of T-cell ALL, forexample, tend to suck upmethotrexate less avidly thanthose of B-cell ALL, so higherdoses of this drug are needed toeffectively treat patients with T-cell ALL. Other patients haveleukemia subtypes that are soresistant to any chemotherapythat doctors take the more drasticmeasure of performing bone mar-row transplants on them, as thisrisky procedure is their only hopefor a cure. In contrast, some sub-types of B-cell leukemia easilysuccumb to chemotherapy inchildren, who therefore requireless than the two or three years ofchemotherapy that is standard forother types of leukemia.

Detecting lurkingleukemiaWhen to stop standardchemotherapy is actually one ofthe more thorny decisions doctorshave to make when treatingleukemia patients. If they admin-ister a group of highly toxicdrugs longer than is necessary,they could subject their patientsto life-threatening side effects.But if they stop too soon, theleukemia could return with avengeance. What these doctorsneed is a reliable way to detectany lingering leukemia cells intheir patients’ bone marrow.

For example, traditionallypatients with ALL were consid-ered to be relatively free ofleukemia if microscopic exami-nation of their bone marrowrevealed a normal number ofimmature B or T cells—no morethan five cells per hundred.Doctors would often stopchemotherapy in patients oncethey reached this goal. But theleukemia would quickly comeback in many patients who satis-fied such crude criteria as each ofthese patients could still harboras many as a billion leukemiacells in their bodies.

In contrast to this insensitivemicroscopic means for detectingleukemia cells in the bone mar-row, flow cytometry can detectthe elusive “needle in thehaystack.” Because doctors canoften use this procedure to detectthe distinctive cell surface signa-tures of their patients’ leukemiacells, they can pinpoint a singleleukemia cell in a sea of ten


thousand normal white bloodcells. If such leukemia cells aredetected after a bout of treatment,then the patient usually receivesanother round of more aggressivechemotherapy.

Greater sensitivity in the detec-tion of leukemia cells has alsocome from a technique known aspolymerase chain reaction (PCR).This revolutionary technique wasfirst conceived of in 1985 by thebiochemist Kary Mullis of asmall California biotechnologycompany. (See the Breakthroughsin Bioscience article on PCRcalled “The Polymerase ChainReaction,” which can be foundonline at http://www.faseb.org/opar/break/).

The PCR technique that Mulliscreated does its molecular detec-tive work by using genetic probesthat seek out a specific geneticsequence. Bacterial enzymes are

then used to copy the desiredsequence repetitively until it isabundant enough to be detectedby various laboratory equipment.

PCR has been used to revealthe specific genetic flaws har-bored by leukemia cells and candetect a single leukemia cellamong nearly a million normalbone marrow cells. Like flowcytometry, PCR can be used tofine-tune diagnosis and detectleukemia cells that remain aftertreatment. This allows doctors tocustom tailor leukemia therapy soit is a better fit for the types ofleukemia their patients have.

Indeed, the ability to use PCR,translocation analyses, or flowcytometry to home in on the pre-cise defects that trigger variousleukemias is a fitting end to amedical detective story that beganwith the crude observations ofVirchow about 150 years ago.

Thanks to a succession of curious scientists from a diversearray of fields, including labora-tory researchers as well as clini-cians, leukemia has gone frombeing a mysterious disease, tobeing a group of well-defineddisorders. By zooming in fromthe gross abnormalities of theseleukemias to leukemia cells’molecular fatal flaws, theseresearchers, from the bench tothe bedside, are gathering infor-mation that is blossoming intocures for many afflicted withthese different types of bone marrow cancers. Because of theircombined efforts, leukemia wardshave gone from being the lastway stations for a fast-approach-ing death train, to being depots of hope—places where doctorscan often ensure that children andother leukemia patients have thelong lives they deserve.

BiographiesMargie Patlak writes about biomedical research and health from the Philadelphiaregion. She has written for Discover, Glamour, Physician’s Weekly, ConsumerReports on Health, The Washington Post, the Los Angeles Times, the Dallas MorningNews, and numerous other publications. She also writes frequently for the NationalInstitutes of Health and the National Academy of Sciences. This is her second articlefor the Breakthroughs in Bioscience series.

Alan S. Rabson, M.D., is the acting director of the National Cancer Institute atthe National Institutes of Health. He came to the NIH in 1955, as a resident in patho-logic anatomy. In 1975, he was named director of NCI’s Division of Cancer Biology,Diagnosis, and Centers, where he served until his appointment as the institute’sdeputy director in 1995. Rabson holds clinical professorships in pathology atGeorgetown University Medical Center and The George Washington University inWashington, D.C., and at the Uniformed Services University of the Health Sciencesin Bethesda, Md. In 1987, Rabson became a member of the Institute of Medicine.

ReferencesFreireich E.J. 1997. The road to the cure of acute lymphoblastic leukemia: a personalperspective. Oncology;54(4):265-9

Jennings, C.D. & Foon, K.A. 1997. Flow cytometry: Recent Advances in Diagnosisand Monitoring of Leukemia. Cancer Investigation, 15(4), 384-399.

Piller, G. 1993. The history of leukemia: a personal perspective. BloodCells;19(3):521-9; discussion 530-5.

Rowley, J.D. 1999. The role of chromosome translocations in leukemogenesis. SeminHematol; 36(Suppl 7): 59-72.

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