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CCR connections

v O LUME 2, n O . 1 | 2008

Joining Forces

Complementary approaches are piecing together the

mysteries of chromosomal translocations and cancer

CENTER FOR CANCER RESEARCH ccr.cancer.gov

U.S. DEPARTMENT 

OF HEALTH AND 

HUMAN SERVICES

National Institutes 

of Health
Center for Cancer Research

National Cancer Institute | National Institutes of Health | Building 31 – Room 3A11 | 31 Center Drive | Bethesda, Md. 20892
Web sites with More Information about CCR

Center for Cancer Research

http://ccr.cancer.gov

Office of the Director

http://ccr.cancer.gov/about/default.asp

Our News

http://ccr.ncifcrf.gov/news/default.aspx

Office of Training and Education

http://ccr.nci.nih.gov/careers/office_training_education.asp

Patient Information on Cancer and Clinical Trials

Open NCI Clinical Trials

http://www.cancer.gov/clinicaltrials

How to Refer a Patient

http://bethesdatrials.cancer.gov/professionals/refer.asp

NCI Cancer Information Service

http://cis.nci.nih.gov

1-800-4-CANCER (1-800-422-6237)

Understanding Cancer Series

http://www.cancer.gov/cancertopics/understandingcancer

Clinical Cancer Trials in Bethesda, Md.

http://bethesdatrials.cancer.gov

Additional Links

National Cancer Institute (NCI)

http://www.cancer.gov

Working at NCI

http://www.cancer.gov/aboutnci/working

National Institutes of Health (NIH)

http://www.nih.gov
NIH Publication No. 07-6211

July 2008
ccr connections | v OLUME 2, N O . 1 | 2008 1

Table of Contents

03 A Different Kind of Cancer Center, a Unique Collaborative Team

NEWS

04 Reverse Transcriptase: When Function Follows Direction

05 Taking Time-Lapse Genomic Snapshots of Cervical Cancer

06 New Clues to Blocking Metastasis in Sarcoma

07 Recent CCR Awards

08 Transposon, Regulate Thyself

09 Finding What’s Real, Functionally Speaking

10 A Cancer Center Shines in Jordan

11 Staff News at CCR

F EATURES

12 Joining Forces

16 One Man’s Junk DNA Is Another Man’s Treasure: 

MicroRNAs and the Next Big Thing in Cancer Prognosis

22 A Better Immunotoxin

26 No Longer Skimming the Surface of Skin Cancer 

COMMENTARY

30 A Series of Fortunate Events

I N THE CLINIC

32 Combating HIV, One Tumor at a Time

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FEATURE

One Man’s Junk DNA 

Is Another Man’s Treasure:

MicroRNAs and the 

Next Big Thing in Cancer

Prognosis

26

FEATURE

No Longer Skimming the

Surface of Skin Cancer 

FEATURE

Joining Forces

FEATURE

A Better Immunotoxin
2 ccr connections | v OLUME 2, N O . 1 | 2008

The mission of the CCR is:

To inform and empower the

entire cancer research community by 

making breakthrough discoveries

in basic and clinical cancer research

and by developing them into novel

therapeutic interventions for adults

and children afflicted with cancer or

infected with HIV.

Contributors:

L.M. Bennett, Ph.D.

D. Kerrigan, M.S.

S. Fox, B.A., B.S.W.

Rhoda Baer Photography

J. Kelly

Vanchieri Communications

Designed and Produced by:

Feinstein Kean Healthcare
ccr connections | v OLUME 2, N O . 1 | 2008 3

The National Cancer Institute’s own clinical

cancer program, under the direction of the

Center for Cancer Research (CCR), sits

squarely at this intersection of exploding

basic cancer biology knowledge and new

cancer treatments. Although we may not

have the same name recognition as other

national cancer centers, which are primarily

large volume, full-service cancer treatment

hospitals and clinics, we are in fact the

largest cancer-focused clinical research center in the world. But we are not a traditional

cancer care center that happens to do

research: Our sole mission is to perform

patient-intensive clinical research aimed at

translating the explosion of new scientific

insights into new approaches to prevent,

diagnose, and treat cancer.

One of our greatest strengths is our

unmatched broad integration of basic and

clinical scientists. This combination is 

particularly powerful at NCI, where basic

laboratories adjoin clinical wards. Thanks 

to this tight collaboration of researchers

across disciplines and settings, we are

uniquely able to knit together insights 

from preclinical disease models with 

science-based approaches to human clinical

treatment. This ability is especially true in

understudied cancers, although our findings often have relevance to many cancers

and even other diseases. Our collaborative

model allows us to identify new cancer therapies, both single agents and combinations,

and bring them rapidly through early 

development and clinical testing and, if 

successful, partner with extramural collaborators to ensure that they are quickly

brought to widespread trials and hopefully

into clinical practice. Our structure also

uniquely enables us to find novel ways to

detect cancer early and even prevent it,

largely through building powerful new 

imaging technologies that allow us to “see”

cancer even in its initial stages.

But perhaps the most important 

collaborative partners of all, the ones who

truly make the NCI cancer clinical research

center unique from all others, are the

patients who come to us. They come here

seeking to actively participate in research

with our basic and clinical scientists to drive

understanding of their cancer and, ultimately, to find an effective treatment—if not for

them, then for others. It is hard to describe

the selflessness—the nobility—of those

who participate in the NCI clinical trials that

are performed at the National Institutes 

of Health Clinical Research Center here in

Bethesda, Md. We are proud to work with

them every day, and their hope and focus

underlie ours. If—when—we succeed in our

mission, it is clear that the lion’s share of

the credit must go to them.

Lee Helman, M.D.

Scientific Director for Clinical Research

Center for Cancer Research

A Different Kind of Cancer Center, a

Unique Collaborative Team

The sheer complexity of human biology, both normal

and disease-related, continues to amaze and often baffle

us as we struggle to unravel it so that we can reduce

the burden of suffering and death that results from 

cancer. Yet understanding cancer biology is only half

the battle: We must actively and rapidly translate that

knowledge into clinically meaningful applications.

Reaching that goal requires a strategic interdisciplinary

and multidisciplinary effort to bridge critical basic 

science with clinical care.

editorial

(Photo: R. Baer)
4 ccr connections | v OLUME 2, N O . 1 | 2008

While the structural sites of RT’s polymerase

and RNase H activities are well known, the

parameters that determine when and how

each site is engaged are not. How does RT

decide whether to enter polymerase or RNase

H mode? 

Using combinations of DNA- or RNAbased primers and templates, the groups of

Harvard University’s Xiaowei Zhuang, Ph.D.,

and Stuart Le Grice, Ph.D., Head of CCR’s RT

Biochemistry Section, have found that RT‘s

function is controlled by its orientation. 

In this system, when presented with 

a DNA primer bound to an RNA or DNA 

template—a scenario analogous to the 

production of a cDNA or vDNA strand—RT

positions itself in a way that favors its polymerase site. However, when RT finds a short

RNA primer bound to a longer stretch of

DNA—which to the enzyme looks like a 

newly transcribed cDNA strand bound to 

its original vRNA template—it binds in the

opposite orientation, promoting its RNAtemplate-degrading RNase H activity.

Remarkably, the researchers also found

that when RT sees a substrate that is

amenable to both of its functions, such as

polypurine tracts (vRNA sequences that in

nature serve as primers for reverse transcription of the HIV genome), the protein flips

spontaneously between orientations, potentially helping it maximize the efficiency with

which it transcribes and degrades the viral

RNA. Exposure to non-nucleoside RT

inhibitors (NNRTIs)—a class of potent and

clinically approved anti-HIV drugs that bind

to RT’s polymerase site—significantly altered

RT’s flipping behavior, forcing the enzyme to

adopt its RNase H-promoting orientation. 

These data, published in the May 8,

2008, issue of the journal Nature, provide significant insights into the structural biology

underlying the reverse transcription process

in HIV and lend new evidence to the observation that NNRTIs can, in certain circumstances,

prevent the synthesis of DNA by reversing

enzyme orientation.

Reverse Transcriptase: When

Function Follows Direction

(Image: S. Le Grice, CCR)

Getting into a host cell’s genome can be a tough job for HIV. Before it 

can be integrated, the virus’ single-stranded, RNA-based genome must

undergo a complete transformation into double-stranded DNA, a multistep

process that holds much room for error. This transformation is where HIV’s

multifunctional reverse transcriptase (RT) comes into play. RT does three

things: transcribes the viral RNA (vRNA) into a single, complementary

DNA (cDNA) strand; destroys the original vRNA; and uses the new

cDNA as a template for a complete double-stranded viral DNA (vDNA).

These three activities require that RT behave in two seemingly contradictory

ways: as a polymerase and as an RNase H.

NEWS

The direction in which HIV’s reverse

transcriptase binds to the viral

genome determines whether the

enzyme reads the RNA or degrades 

it. This knowledge has shed new 

light on the mechanisms by which

antiviral drugs called non-nucleoside

RT inhibitors act against HIV. 
ccr connections | v OLUME 2, N O . 1 | 2008 5

Not all women who contract HPV develop

cervical cancer. In many, the series of precancerous changes triggered by the virus, called

cervical intraepithelial neoplasia (CIN), 

progresses to a certain point and simply

stops. In those women who do develop 

cancer, the process can take mere months or

go on for years. 

Answering the how and why would be

easier if researchers had a time-lapse series

of genomic snapshots spanning the cellular

progression from viral infection to invasive

malignancy. In the August 1, 2007, issue of

Cancer Research, a multi-institutional team of

Taking Time-Lapse Genomic

Snapshots of Cervical Cancer

Striking shifts in gene expression at different time points in the process of cervical cancer development, from human papillomavirus

(HPV) infection to invasive carcinoma, may help reveal how HPV goads cervical cells into becoming cancerous and why the process of

carcinogenesis varies so much from woman to woman. 

NEWS

(Image: J. Kelly)

scientists including CCR’s David Gius, M.D.,

Ph.D., Head of the Molecular Radiation

Oncology Section, unveiled just such a 

comprehensive photo series. Working with

Janet Rader, M.D., and colleagues at

Washington University School of Medicine

in St. Louis, Mo., Gius examined gene

expression within the cervical epithelium

(the layer of cells that HPV infects and where

cervical cancer originates) and the underlying stroma (normal cells that form a

developing tumor’s microenvironment) 

at different histological time points in

cervical cancer development.

The team found striking shifts in gene

expression that correlated closely with the

progressive changes seen under the microscope as the cervical epithelium transforms.

Their results suggest three distinct genomic

phases in the development of cervical cancer. First, an immunosuppressive phase,

characterized by genes linked to the establishment of HPV infection and immune 

system evasion. Second, a pro-angiogenic

phase, where the precancerous cells “talk” to

the nearby stroma and encourage the production of factors fostering blood vessel

growth, possibly corresponding to the

“angiogenic switch” proposed a decade ago

by the late Judah Folkman, M.D. Finally, a

pro-invasive phase, associated with genes

that promote the breakdown and invasion of

neighboring healthy tissues.

Together these results constitute an in

vivo model of cervical cancer development

that both provides a window on the evolution

of cervical cancer and highlights a number 

of genes with prognostic and therapeutic

potential.

Dividing cells

in basal layer

Normal Very mild/

mild dysplasia

Moderate

dysplasia

Severe

dysplasia

In situ

carcinoma

Invasive

carcinoma

CIN 1 CIN 2 CIN 3 SCCA

Proliferative/

immune suppression

genomic signature

Pro-angiogenic

stromal-epithelial

genomic signature

Pro-invasive

genomic signature

SMARCA4

Loss of contact

with BM

results in

Anoikis

Inhibition

Anoikis

Microvesicle

Bleb

CELL

DEATH

Cell

Cycle Modified Desmosome

Modified

Desmosome

Pro-Apoptosis

Genes

IFNα

IFNAR1

IFNAR1

P2RX7

STAT1 STAT2

IF

STAT 1/2 NAR2

IFNAR2

ITGAV

β3

Tyk2

HINT1 MITF

E7

IL1

KAL1

MAP2K7

TBX19

DSG3

DSG3

αMSH

FGFR1

FGFR1

VEGF

TWEAK

MMP14 PRO-MMP2

TNFRSF12A

IL1RN

ACAA1

ACAA1

EMP1 P2RX7

EMP1

IL1RN

ILR1

E6

E6

Rb

Rb

E7

Apoptosis

APOPTOSIS

ANGIOGENESIS

Apoptosis Virion

Assembly

HPV

Components

MAST CELL

PROLIFERATION

Jak1

POMC

HIF1α

Jak1

mMCP4

IFNα

IFN

CDKN2A CDKN2A NOMO2 NF-κβ

NF-κB

MMP14

CDK4

CDK4

FGF2

VEGF FGF2 FGF2

SWI/SNF

complex

Tyk2

Cyclin D1

CENPE

CENPF KIF23

RXR 9-cis

RA

Smad

2/3

HSPG

Smad

2/3 Smad2/3

Nodal MMP2

PPARα ligand PPARα

Peroxisome

CIN 1 CIN 2 CIN 3 SCCA

Proliferative/immune suppression

genomic signature

Pro-angiogenic stromalepithelial genomic signaturePro-invasive

genomic signature

HIF1α

ALK7

E2F Rb

E2F

MC1R

Rb

E2F

E2F

E2F

Cyclin D1

MMP3

MMP3

MMP2

2-3 mm depth 2-3 mm depth

VEGF

SUMO1

HSPG

KAL1

BRWD1

1See “A Victory against Cancer: 25 Years in the Making,” CCR Connections, Vol. 1, No. 1.

Researchers and physicians have known for over a quarter of a century

that human papillomavirus (HPV) infection underlies the vast majority

of cervical cancers. While the approval of the first HPV vaccine1 in

2006 was a breakthrough in cervical cancer prevention, important

questions remain. How does the virus fuel cervical cancer development

and why do the developmental steps vary from woman to woman? 
6 ccr connections | v OLUME 2, N O . 1 | 2008

Cancer’s wanderlust can be its most fatal

feature. Metastasis—the spread of cancer

cells to sites far from the original tumor—is

the cause of death for approximately 90 percent of cancer patients. It is not a simple,

one-step process: To metastasize, cancer

cells must escape the original tumor, enter

and exit the bloodstream or lymphatic 

system, and establish themselves in a new

target organ or tissue. All told, the process

requires the activation of several genes and

the deactivation of several others.

Findings from a team of CCR

researchers suggest that for at least some

sarcomas (a group of cancers of the connective and supportive tissues, such as

bone, cartilage, fat, muscle, or blood vessels), cancer cells may get the green light to

metastasize not because of genomic

changes but because a protein important

for suppressing this process is tagged for

destruction. This insight has come about

because the CCR researchers—led by Allan

M. Weissman, M.D., Chief of the Laboratory

of Protein Dynamics and Signaling, and

Chand Khanna, D.V.M., Ph.D., of the

Pediatric Oncology Branch—chose to study

not genomic mechanisms of metastasis but

rather the fate of the proteins produced

from these genes. 

Weissman and Khanna focused their

attention on gp78, one of many proteins

whose job it is to mark other proteins with

a small protein called ubiquitin. The cell

normally uses this molecular tag to flag

excess or damaged proteins for destruction

by a complex cellular machine called the

proteasome. 

Using RNA interference (RNAi) in an

animal model of sarcoma, the researchers

turned down gp78 expression, finding that

metastasis was similarly reduced. Looking

at gp78’s interactions, they found that it

tags a protein called KAI1—one of about 

10 known metastasis suppressors—for

degradation by the proteasome. In their laboratory sarcoma models, the team found

that reducing levels of gp78 caused KAI1

levels protein to rise and the survival of

metastatic sarcoma cells to diminish.

Subsequently, turning gp78 expression back

up reversed the situation: KAI1 was tagged

more for destruction more frequently, and

metastatic cancer cells with the rejuvenated

gp78 expression had better survival. 

To establish the clinical relevance of

their findings, the scientists looked at

archived clinical samples of sarcoma

tumors. There, too, they found evidence for

an inverse relationship between the two

proteins: When levels of gp78 were low,

KAI1 levels were high, and vice versa. 

The structure of gp78 makes it a

potential candidate for targeted therapeutics. Spurred on by the findings, the CCR

team, as well as other scientists, is now

beginning to look for similar relationships

between gp78 and metastasis in other cancers. The results also open the door to

exploring the use of available proteasome

inhibitors, such as bortezomib (Velcade®),

to treat metastatic cancers. Bortezomib is

already successfully used to treat the blood

cancer multiple myeloma and has been

widely studied in other cancers.

Suppressing a metastasis-suppressing protein, called KAI1, by tagging it for

degradation by the proteasome may be one factor in a sarcoma cell’s development of the ability to spread. 

New Clues 

to Blocking

Metastasis

in Sarcoma

NEWS

(Image: L. Liotta, CCR)
ccr connections | v OLUME 2, N O . 1 | 2008 7

American Society for Public Service 

and National Academy of Public

Administration National Public 

Service Award

~ For excellence in public service

Peter M. Blumberg, Ph.D.

Laboratory of Cancer Biology 

and Genetics

American Association of Immunologists 

Public Service Award 

~ For advancing immunological 

research related to aging

Richard J. Hodes, M.D.

Experimental Immunology Branch

American Society of Investigative 

Pathology Chugai Award

~ For excellence in mentoring and 

scholarship

Elaine S. Jaffe, M.D.

Laboratory of Pathology

American Medical Association 

Dr. Nathan Davis Award 

~ For outstanding government service

W. Marston Linehan, M.D. 

Urologic Oncology Branch

Service to America Medal

~ For selection as Federal Employee 

of the Year

Novartis Prize for Immunology

~ For excellence in immunology 

research

Douglas R. Lowy, M.D.

John T. Schiller, Ph.D.

Laboratory of Cellular Oncology

American Cancer Society's Medal 

of Honor 

~ For basic research

Eugene J. Van Scott Award and Phillip

Frost Leadership Lecture

~ For innovative therapy of the skin

Douglas R. Lowy, M.D.

Laboratory of Cellular Oncology 

Society for Melanoma Research Senior

Researcher Award

~ For development of murine 

melanoma models

Glenn Merlino, Ph.D.

Laboratory of Cancer Biology 

and Genetics 

Twelfth Annual Keio University Medical 

Science Prize, Japan

~ For development of AIDS drugs

Hiroaki Mitsuya, M.D., Ph.D. 

HIV and AIDS Malignancy Branch

Weizmann Institute of Science Sergio 

Lombroso Award

American Society of Clinical Oncology

Statesman Award

Abbott Laboratories Award in Clinical 

and Diagnostic Immunology

~ For distinguished achievement in 

cancer research

Steven A. Rosenberg, M.D., Ph.D.

Surgery Branch 

American Association of ImmunologistsDana Foundation Award in Human

Immunology Research

~ For significant and sustained 

achievement in immunology research

Thomas A. Waldmann, M.D. 

Metabolism Branch 

Academy of Sciences of the Czech Republic

Jaroslav Heyrovsky Honorary Medal for

Merit in Chemical Sciences

~ For contributions to the studies of

protein structure

Alexander Wlodawer, Ph.D.

Macromolecular Crystallography

Laboratory

Stanford Medical Research 

Scholarship Award

~ To fund research and training in 

Tumor Immunology and Biology

Jorge Caballero, B.S. 

Laboratory of Tumor Immunology 

and Biology

NEWS

Recent CCR Awards

CCR Summer Student Was an Intel Semifinalist

Sappho Z. Gilbert, a summer student in Maria Tsokos, M.D.’s Pediatric Tumor

Biology and Ultrastructural Pathology Section of the Laboratory of Pathology, was

one of 40 finalists in the highly competitive Intel International Science and

Engineering Fair for 2008. This competition for high school students awards

approximately $1 million in scholarships each year and recognizes outstanding

students nationally. Gilbert’s project was entitled, “Survivin Correlates with

Prognosis and Inhibits Drug-induced Apoptosis in Ewing Sarcoma Family Tumors.”

(Photo: NIH)
8 ccr connections | v OLUME 2, N O . 1 | 2008

Adding to the frenzy are genes that just cannot sit still. Called transposable elements

or transposons, these “jumping genes”

move about the genome with abandon.

Generally considered to be the remains of

ancient viruses that merged into our DNA

far back in our evolutionary past, transposons exist in two distinct classes: DNA

transposons and RNA-based retrotransposons.

With their ability to randomly cut- or

copy-and-paste themselves into just about

any genomic location, transposons can be

both boon and bane. They can increase

genome size and complexity, providing 

fertile ground for the development and evolution of new traits. Or they can wreak

genomic havoc by disrupting genes critical

for normal cellular functions, such as proliferation and apoptosis. 

Because they can have such widespread and powerful effects, the genome

needs to keep the activity of transposons

under tight control. In the January 24, 2008,

issue of the journal Nature, Laboratory 

of Biochemistry and Molecular Biology

Senior Investigator Shiv Grewal, Ph.D.,

Postdoctoral Fellow Hugh Cam, Ph.D., and

their colleagues reported the results of

studies in yeast revealing the existence of a

powerful mechanism by which the genome

may regulate how and where transposons

move and act. 

This surveillance system centers on

the action of a group of proteins homologous to a human protein family called the

CENP-B family. According to the Grewal

team’s results, the CENP-B proteins—themselves derived from the remnants of past

transposons—seek out and silence a group

of retrotransposons called the Tf2 family.

The CENP-Bs also appear to be able to corral these retrotransposons into clusters,

called Tf bodies, which may facilitate the

proteins’ surveillance tasks and also have

wider implications for genome organization,

gene regulation, and response to environmental stresses.

Taken together, the scientists’ results

suggest that the genome has, over time,

tamed some of these “jumping genes” and

redirected them into roles where they 

regulate their own kin, thereby helping to

maintain genome integrity. 

By silencing and clustering Tf2

retrotransposons (green), proteins

of the CENP-B family (red), themselves the remnants of ancient

transposons, regulate the movement and placement of these

mobile genetic elements and help

maintain the organization and

integrity of the genome. 

The neighborhood around the

genome is not a quiet place. Rather,

it has all of the activity of a city at

rush hour: Chromosomes ravel 

and unravel, DNA unzips while

proteins zoom in for transcription or

replication, and RNAs zoom out for 

translation into proteins. 

Transposon, Regulate Thyself

NEWS

(Image: S. Grewal, CCR)
ccr connections | v OLUME 2, N O .1 | 2008 9

Such is the case for the DNA repair genes

BRCA1 and BRCA2. Mutations in these

genes can significantly increase a woman’s

risk of developing breast cancer. While

sequencing-based tests can help women

assess their risk and make appropriate

decisions about preventive treatment, the

functional impacts of nearly 1,900 identified BRCA1 and BRCA2 mutations on the

genes’ protein products remain unclear.

Tying mutations to functions would help

improve breast cancer risk assessments;

however, such functional studies are both

labor- and resource-intensive.

CCR’s Shyam Sharan, Ph.D., Head of

the Mouse Genetics Program’s Genetics 

of Cancer Susceptibility Section, and

Research Fellow Sergey Kuznetsov, Ph.D.,

have developed an in vitro functional assay

system capable of accurately and relatively

rapidly measuring the effects of BRCA2

mutations. The pair, together with Pentao

Liu, Ph.D. (a former CCR Research Fellow

now at the Wellcome Sanger Trust

Institute), published a report on their system in the July, 2008, issue of Nature Medicine.

The functional consequences of mutations in BRCA2 (represented here in a 

multispectral karyotype displaying a translocation between chromosomes 4 

and 13) and other genes can now be better and more rapidly understood thanks

to a new in vitro validation system based on mouse embryonic stem cells.

Finding What’s Real,

Functionally Speaking

Not all mutations are created equal. Where some can be silent, having no

discernable effect on a cell, tissue, or organ, others can be profoundly 

disruptive.1 Deciding which category a mutation fits into is often the subject

of significant amounts of experimental work and debate, particularly 

when the gene or genes in question could potentially raise or lower the risks

of developing diseases like cancer.

NEWS

(Image: S. Burkett, CCR)

The system measures a mutation’s

functional impact using a knockoutand-replace strategy. The researchers

replaced both normal Brca2 gene copies in

mouse embryonic stem cells (ESCs) with

copies of human BRCA2 containing particular mutations of interest. Measurements

of the cells’ growth, survival, and sensitivity

to agents (chemicals, radiation, etc.) that

cause DNA damage indicated which mutations had no effect and which kept BRCA2

from compensating for the loss of Brca2.

Using this system, Sharan, Kuznetsov,

and Liu examined the functional impacts 

of 17 catalogued BRCA2 mutations. Their

findings validated the known effects of 13 of

the mutations and gave the first evidence 

of the impacts of four others whose functional impacts had not been characterized 

previously. The researchers noted that 

with their system, between three and five

mutations can be assessed on a two- to

three-month timeframe, and the system

could potentially be used with any gene

mutation that causes a phenotype in mouse

ESCs, making this system a useful tool for

genetic counselors. They caution, though,

that the system needs to be validated further

before it is ready for clinical application.

1

For more information, see “Silent No More,” CCR Connections,

Vol. 1, No. 1.
1 0 ccr connections | v OLUME 2, N O . 1 | 2008

NEWS

In 2002, this medical center, located in

Amman, Jordan, and called the Al-Amal

(“Hope”) Center, had significant quality and

safety problems. A large percentage of its

patients did not know they had cancer. It was

not even called a cancer center. 

That year, the monarch, King Abdullah,

asked NCI to help his country develop a 

high quality cancer center. Khleif—an NCI

oncologist, cancer vaccine specialist, and

Palestinian-American with a medical degree

from the University of Jordan—moved to

Amman to become KHCC’s Director General

and CEO. 

Today, remade at his hands and

renamed for the late King Hussein, KHCC is

the only comprehensive cancer center in the

Middle East. 

To transform the center, Khleif radically

changed its culture and organization. The

most important thing, he said, was implementing quality measures and building

teams among the staff, thereby engendering

a sense of commitment to the center’s mission. “It became something of a jewel, a

source of pride for the country,” he said.

Khleif’s leadership motivated KHCC

staff to “make significant sacrifices that went

beyond their own self-interest,” according to

a case study by Duke University researchers

published in November 2007 in Globalization

and Health. “There was a sense of purpose or

vision-driven efforts to attend to the needs

of patients and mid- and lower-level hospital

staff...in an effort to rapidly raise the standard of care at KHCC.” 

The results, in terms of morale, performance, and outcomes, have surpassed all

expectations. Staff retention is high (“They

feel they are accomplishing things. They’ve

shown they can improve healthcare,” noted

Khleif). KHCC residents now outrank those

of top-tier American medical centers on 

the American College of Physicians’ annual

in-service exam (Jordan is one of only six

countries outside of the U.S. and Canada

that participate in this evaluation of resident

performance). And in February 2006, KHCC

received accreditation from the Joint

Commission on International Accreditation

(JCIA), a division of the U.S.-based Joint

Commission on the Accreditation of

Healthcare Organizations (JCAHO). 

“We built a Western-style medical

institution, introduced quality management

in healthcare, and got JCIA accreditation, a

very important accomplishment worldwide,”

Khleif said. “We established the first support

groups, the first palliative care program, 

the first early detection unit, and the first 

smoking cessation clinic in the country.

These actions changed the whole landscape

of healthcare in the country.” 

Khleif returned to CCR and his laboratory in the Cancer Vaccine Branch the day

after KHCC received JCIA accreditation.

But his ties to Jordan remain strong. King

Abdullah has asked for additional help to

build a cancer and biotechnology institute.

Khleif is again at the helm, traveling

between Bethesda and Amman to guide the

birth of a new hospital and laboratories for

basic and translational research, a CCR in

the desert. 

The KHCC treats more than 50 percent of Jordan’s cancer patients. About one quarter of

its patients come from outside the country, mainly Syria, Iraq, the Palestinian Authority,

Sudan, Libya, Algeria, and Yemen.

Samir Khleif, M.D.

A Cancer Center Shines in Jordan

The King Hussein Cancer Center (KHCC) is a story of transformation

and purpose-driven aspirations. And CCR’s Samir Khleif, M.D., is a

central reason for its success. 

(Image: Courtesy of S. Khleif)

(Photo: E. Branson)
ccr connections | v OLUME 2, N O . 1 | 2008 1 1

NEWS

Staff News at CCR

(Photo: R. Baer) (Photo: E. Branson) (Photo: M. Spencer) (Photo: M. Spencer) (Photo: Courtesy of P.

Kalab)

(Photo: M. Spencer) (Photo: E. Branson)

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L. Michelle Bennett, Ph.D. 

Bennett has been named Deputy Director of CCR. She trained at the University of Wisconsin and at the

National Institute of Environmental Health Sciences. Since joining CCR in 2002 as Associate Director for

Science, she has administered numerous initiatives, including CCR’s strategic plan and the new collaborative

Centers of Excellence. As Deputy Director, Bennett will focus on strategic planning, program integration, and

scientific communications.

Kevin Camphausen, M.D. 

Camphausen has been appointed Chief of the Radiation Oncology Branch (ROB) where he previously served

as Deputy Branch Chief and Acting Chief. Before joining NCI in July 2001, he studied the interaction of angiogenesis inhibitors and radiotherapy at Harvard Medical School. He envisions the ROB at the cutting edge of

combined modality therapy with treatment strategies that merge radiation and molecular targeted agents. 

J. Carl Oberholtzer, M.D., Ph.D. 

Oberholtzer joins CCR as Chief of the Laboratory of Pathology. Oberholtzer originally came to the NCI in 2006

as Associate Director for Training. Before NCI, he was Associate Professor of Pathology and Laboratory

Medicine, Director of the Division of Neuropathology, and Vice Chair of the Pathology Department at the

University of Pennsylvania. He received his Ph.D. from the University of Pennsylvania and his M.D. from

Jefferson Medical College. 

Itzhak Avital, M.D. 

Avital joins the NIH Clinical Center’s Surgery Branch as a Tenure-Track Investigator. He came to CCR in 2006

to launch a solid-organ cancer stem cell research program. Trained in surgery and surgical oncology at the

Memorial Sloan-Kettering Cancer Center (MSKCC), Avital received his M.D. from New York University’s School

of Medicine. 

Petr Kalab, Ph.D. 

Kalab returns to NIH as a Tenure-Track Investigator in CCR’s Laboratory of Cellular and Molecular Biology. Kalab,

from the University of California at Berkeley, developed molecular imaging tools to study the RanGTP gradient in

live cells. At CCR, he will investigate Ran-regulated mitotic functions and their overlap with misregulated pathways

in cancer.

Udai S. Kammula, M.D. 

Kammula joins the Surgery Branch as a Tenure-Track Investigator. Trained in surgical oncology at MSKCC and

the Surgery Branch of the National Cancer Institute, his clinical work focuses on malignancies of the liver, 

pancreas, and gastrointestinal tract. His research focuses on tumor immunology and immunotherapy 

development. 

King F. Kwong, M.D., FACS 

Kwong recently joined CCR’s Thoracic Oncology Section as a Tenure-Track Scientist from the University of

Maryland. His CCR research will focus on apoptosis in thoracic malignancies. Kwong received his M.D. from

The George Washington University (GWU) in Washington, D.C., and he received training at both GWU and

Washington University in St. Louis, Mo.

Newly Tenured

CCR Scientists

Mirit I. Aladjem, Ph.D. 

Laboratory of Molecular

Pharmacology 

Javed Khan, M.D. 

Pediatric Oncology Branch 

Jairaj Acharya, Ph.D. 

Laboratory of Cell and

Developmental Signaling
1 2 ccr connections | v OLUME 2, N O . 1 | 2008

It is now known that this specific translocation, found in 95 percent of patients with

CML, fuses a proto-oncogene (a normal

gene with oncogenic potential) on chromosome 9 (c-ABL) to a site on chromosome 22

known as a breakpoint cluster region. This

hybrid oncogene, BCR-ABL, produces a 

constantly activated mutant protein (BCRABL), which wreaks the genomic havoc in the

cell that ultimately causes CML but which also

is the target of imatinib mesylate (Gleevec®),

the first FDA-approved treatment to target a

translocation-specific fusion protein.

Since the 1970s, chromosomal

translocations have been associated with

several types of blood cancers, and they are

recognized increasingly as key players in

solid tumors as well. Today, cytogenetic testing is performed frequently in the clinic to

determine which chromosomal translocations are present in patient samples, helping

facilitate diagnosis as well as treatment

planning. But although we have come a long

way in 50 years, we have yet to fully understand the molecular mechanisms behind

these deadly chromosomal rearrangements

and why they happen so often in the same

chromosomal locations. Instead of treating

the resulting cancer, can we prevent them

from occurring in the first place?

Researchers in the Center of Excellence in

Chromosome Biology at CCR are putting

together answers to these questions.

Joining Forces

Complementary approaches are piecing together the mysteries of

chromosomal translocations and cancer

f eature

(Image: A. Nussenzweig, CCR)

It has been almost 50 years since a “minute chromosome” was first 

identified in patients with chronic myelogenous leukemia (CML). This

genetic abnormality, named the Philadelphia chromosome for where it

was discovered, was the first genetic defect linked to cancer (Figure 1).

Investigators initially believed that the Philadelphia chromosome resulted

from the loss of genetic material. However, advances in cytogenetics over

the next decade made it possible to view the true nature of this abnormality

—the genetic material “missing” from chromosome 22 was not lost but

“translocated” to chromosome 9.
ccr connections | v OLUME 2, N O . 1 | 2008 1 3

Taking a Global View

Tom Misteli, Ph.D., Head of the Cell Biology

of Genomes Group in CCR’s Laboratory of

Receptor Biology and Gene Expression,

arrived at NCI nine years ago with the task of

building an imaging program. Within a few

years, he gradually began to apply in vivo

imaging techniques to chromosome biology

and specifically to understanding how

genome organization affects genome regulation (see “The Right Place at the Right

Time”). He and his CCR colleagues are now

using high resolution microscopy, live-cell

imaging, and computer simulation to study

the positioning of entire chromosomes and

particular gene loci within the nucleus in

order to understand how these arrangements change during normal and aberrant

physiological processes (Figure 2). 

Since the 1970s, 

chromosomal 

translocations have

been associated with

several types of

blood cancers, and

they are recognized

increasingly as key

players in solid

tumors as well. 

Figure 1: The Philadelphia chromosome

—the result of a translocation between

chromosomes 9 and 22 (circles)—is

often found in the cells of patients with

chronic myelogenous leukemia. 

(Image: Feinstein Kean Healthcare)

freedom of movement. To better understand these findings, Misteli teamed up

with Andre Nussenzweig, Ph.D., Head of 

the Molecular Recombination Section at 

CCR’s Experimental Immunology Branch.

Nussenzweig and his colleagues (see

“Making the Right Connections”) explore

chromosomal translocations at the molecular level—an approach that complements

Misteli’s “macro” view of chromosome biology—to focus on the cellular mechanisms

of genomic stability and how defects in

DNA repair and cell cycle checkpoints 

lead to chromosomal translocations and

malignancies. 

Misteli and Nussenzweig designed

an experimental system that allowed them

to introduce a double-strand break within

the genome at will and then visualize the

fate of each of the damaged DNA ends in

living cells in real time. They found that

while the broken chromosome ends do 

separate slightly from each other, their

long-range motion is significantly constrained. This positional stability depends

upon the presence of a specific DNA 

binding protein known as Ku80, a component of the nonhomologous end-joining

(NHEJ) DNA-repair pathway. Data from

Ku80 knockout mice generated by the

Nussenzweig lab demonstrate that Ku80 

is important for maintaining genomic 

stability—it forms an asymmetric ring

around the two broken ends to align them

for repair. Several follow-up studies are

being conducted to further characterize this

protein and the role it plays in DNA repair

and chromosomal translocations.

Far from being randomly scattered

around the nucleus, chromosomes, subchromosomal domains, and individual

genes are nonrandomly organized into discrete territories, or neighborhoods, within

the nucleus. Although these entities may

have preferred localizations, the patterns

can change in response to the cellular environment. The distinct territory occupied

varies with cell type, upon cell differentiation, and when cells exit the cell cycle, 

suggesting a link between positioning and

genome function. The positioning is

believed to influence gene expression programs as cells undergo changes throughout

development and differentiation. 

Positioning can determine direct

interactions between genes, which in turn

can regulate gene expression. In naïve Thelper immune cells, for example, when a

specific region on mouse chromosome 11

(TH2) directly interacts with the lfng locus

on chromosome 10, the locus is turned “off.”

When these cells then receive a signal to differentiate, the two regions separate, and the

lfng locus turns back “on.”

Chromosomal translocations occur

when direct physical interactions go wrong,

and these can be deadly. DNA doublestrand breaks occur frequently throughout

the genome during replication or as a result

of DNA-damaging agents like radiation.

What remains unclear is how the broken

ends of two different chromosomes—which

should not be allowed to “mingle” under

normal circumstances—assemble and fuse

together, forming a translocation. 

Spatial mapping studies by Misteli’s

laboratory demonstrated that the breakage

sites of several common translocations (e.g.,

Myc-Igh, BCR-ABL, and RAR-PML) are preferentially positioned in close proximity to

each other in normal B lymphocytes prior to

undergoing chromosomal rearrangement.

This observation suggests that proximity

may be a requirement for translocation.

Taking a Closer Look

This proximity requirement suggests that

broken DNA ends do not just flop around

in the nucleus but are fairly limited in their

Chromosomal

translocations occur

when direct physical

interactions go

wrong, and these can

be deadly.

feature
1 4 ccr connections | v OLUME 2, N O . 1 | 2008

feature

Of Recombination and

Translocation

The NHEJ pathway is a key pathway in normal immune cell development through its

involvement in V(D)J recombination. This

normal programmed recombination event

occurs early in the life of developing B 

lymphocytes, resulting in the generation of

cell surface receptors that can accurately

identify a massive diversity of intruders and

mobilize the immune system to respond.

During recombination, the variable (V),

diversity (D), and joining (J) gene segments

are selected randomly and recombined to

ultimately produce an antigen receptor

gene. These receptors can even recognize

microbes that an individual or his/her ancestors have never encountered, which explains

how some individuals are naturally immune

to new infections or viral strains. 

By studying the process of V(D)J

recombination, Nussenzweig and colleagues

hope to gain invaluable insight into the

mechanisms responsible for chromosome

translocations. Aberrant chromosomal

translocations between antigen receptor loci

and proto-oncogenes are a hallmark of lymphoid cancers. These translocations are not

random—they involve only a few oncogenes

with recurrent breakpoints. Mature B-cell

lymphomas, for example, typically involve

fusions of antigen receptor loci with BCL1

(mantle zone lymphoma), BCL2 (follicular

lymphoma), or MYC (Burkitt’s lymphoma). 

V(D)J recombination is initiated by the

RAG-1/2 endonuclease, an enzyme that

introduces specific double-strand breaks

within the VDJ genes. The broken DNA

strands are then fused together via the NHEJ

pathway to form the antigen receptor gene.

The strands are prevented from joining illegitimately by ataxia-telangiectasia mutated

(ATM), a key enzyme that is activated by DNA

double-strand breaks. ATM also prevents the

propagation of cells with broken chromosomes by activating mechanisms that lead

to cell cycle arrest, DNA repair, or apoptosis

(cell death) (Figure 3). Mutations within the

ATM gene result in the rare disorder ataxia

telangiectasia (from which the gene name is

derived), which is characterized by immunodeficiency and predisposition to lymphoid

malignancies caused by chromosomal

translocations near antigen receptor genes. 

Nussenzweig’s group discovered that

ATM is also part of a system that prevents

genetic damage from being passed on to a

cell’s offspring. In a recent study published

in Cell, Nussenzweig and his brother, the

Rockefeller University’s Michel Nussenzwieg,

M.D., Ph.D., showed that when ATM is

absent, chromosomal breaks created during

V(D)J recombination go unrepaired, and

checkpoints that normally prevent the damaged cell from replicating are lost.

Remarkably, the cell divides, matures, and

maintains the breaks, which can persist for

more than five generations in vitro and

approximately two weeks in vivo. Since ATM

is mutated in a number of lymphomas, the

new finding suggests to researchers that the

lymphocytes could have been living with

DNA damage for a long time, and that this

damage likely plays a role in later chromosomal translocations that lead to cancer. This

novel form of “delayed” genomic instability,

which permits more time for long-range

movement of chromosomal breaks, might

Figure 2: The positioning of the 23 pairs

of chromosomes within the nucleus

(here labeled with red, green, and blue

dyes) as a cell goes through the cell

cycle may significantly influence gene

expression. 

Making the Right

Connections

Andre Nussenzweig,

Ph.D. 

Head, Molecular

Recombination

Section, Experimental

Immunology Branch

Andre Nussenzweig takes

a different angle toward understanding how

and why chromosomes break and rejoin and

how they relate to cancer: that of a physicist

(Ph.D. from Yale) and an expert in laser spectrometry. This background has, during his

nearly 10 years with NCI/CCR’s Experimental

Immunology Branch, given him a unique perspective on the mysteries of cell nuclei. “Al

Singer was crazy enough to hire me,” he

joked.

Nussenzweig “learned biology” during

a postdoctoral stint at Memorial SloanKettering Cancer Center, where he studied a

mouse model of defective DNA repair. It was

there that he first teamed up scientifically

with his brother Michel, a Howard Hughes

Medical Institute Investigator at the

Rockefeller University. This familial collaboration continues to this day.

Elsa Callen, Ph.D.

Visiting Fellow

For the past three years in

Nussenzweig’s lab, Elsa

Callen has been working

to understand the possible roles of DNA repair

proteins normally seen in diseases like lymphoma. Before joining NCI, Callen completed

her Ph.D. at the Universitat Autonoma de

Barcelona (Spain) at the end of 2004, where

she studied Fanconi anemia, a devastating

disease that can be caused by a set of genes

involved in DNA repair. This work led her to

seek further understanding of DNA repair

mechanisms in Nussenzweig’s lab.

“Working here has many advantages,”

said Callen, particularly in the availability of

necessary resources and the collaborative

atmosphere. “It makes it really easy to 

develop interesting, challenging, and high

quality projects that would be almost

impossible under other circumstances.”

(Image: T. Misteli, CCR)

(Photo: R. Baer) (Photo: R. Baer)
ccr connections | v OLUME 2, N O . 1 | 2008 1 5

feature

Figure 3: If the protein ATM (blue and

yellow) is mutated, double-strand DNA

breaks can go unrepaired and result in

genomic damage that can be passed on

to daughter cells. If unchecked, this

damage can promote the development 

of lymphomas and the rare disorder

ataxia telangiectasia.

Tom Misteli, Ph.D. 

Head, Cell Biology of Genomes

Group, Laboratory of Receptor

Biology and Gene Expression

Tom Misteli’s focus when he joined NCI was

on setting up a cellular imaging program,

based on his pioneering efforts to visualize

gene expression and protein dynamics in living cells. However, he

soon started looking at the behavior of whole chromosomes within

their nuclei. “I always wanted to do work on chromosome positioning, but it just wasn’t fundable outside of the NIH,” said Misteli. 

His success has attracted numerous research fellows. “We have

10 to 12 people at any time now, all extremely good at what they do,”

said Misteli. “In fact, every postdoc in my lab is better than I ever was.”

Misteli received his Ph.D. from the University of London and

the Imperial Cancer Research Fund, London, United Kingdom.

Karen Meaburn, Ph.D. 

Visiting Fellow

Karen Meaburn has been doing research in

the Misteli lab for the past two and a half

years on understanding chromosome and

gene positioning, particularly in breast 

cancer. Her hope is that her work will help

lead to ways to detect breast cancer at its earliest stages. 

The Right Place at the Right Time

According to Meaburn, being at NCI provides her with both

the support she needs and intellectual stimulation beyond her

immediate area of study. “There is the opportunity to get the

resources to try things out,” she said, “and there is always someone doing something I am interested in.”

Meaburn received her Ph.D. in biological sciences in 2005

from Brunel University in London, United Kingdom.

Evi Soutoglou, Ph.D. 

Visiting Fellow

Evi Soutoglou has spent four years 

at NIH, first as an International Human

Frontiers Science Program Fellow and

now as a Visiting Fellow in the Misteli lab.

She says that imaging work was an

“obvious” choice for her, and that the capabilities and research

interests at NCI attracted her, particularly in working in the

Misteli lab in understanding the stability of chromosome breaks.

“Tom is not afraid to try new things and brings the energy

and resources to help us address difficult but interesting 

questions,” said Soutoglou, who also cites the collaborative

atmosphere provided by the lab and by her colleagues in other

labs as unique aspects of the NCI intramural program.

Soutoglou received her Ph.D. in 2002 from the University of

Crete’s Institute of Molecular Biology and Biotechnology.

contribute to the oncogenic transformation

of mature lymphocytes. 

In a separate study published recently

in Nature, a group of NCI scientists, led by

Raffael Casellas, Misteli, and Nussenzweig,

identified one mechanism by which ATM

protects cells from passing on genetic aberrations to progeny cells. When mouse cells

are exposed to a DNA damaging agent, the

resulting double-strand breaks transiently

inhibit RNA polymerase I (which is required

for the synthesis of ribosomal RNA) via the

ATM pathway. Thus, in response to chromosomal breaks, ATM shuts down ribosomal

gene expression, which ultimately impacts

the assembly of all cellular proteins. 

This new finding suggests new

avenues of research for understanding the

role of translocations in early disease development. Such insights into the structural

and molecular defects that happen in the

earliest stages of chromosome translocations

have perfectly positioned CCR researchers to

develop novel strategies for the early detection of cancer and, perhaps, to even prevent

these genetic abnormalities from occurring

in the first place. 

(Photo: R. Baer) (Photo: M. Spencer)

(Photo: M. Spencer)

(Image: A. Nussenzweig, CCR)
1 6 ccr connections | v OLUME 2, N O . 1 | 2008

Until very recently, scientists thought they had a pretty good basic model

for how cells work. DNA was the storehouse of all information, the blueprint for life. Proteins were the building blocks: the bricks, mortar, and

switches that actually made a living thing and also made it work.

According to the model, while RNA did a good job shuttling the DNA’s

instructions from the nucleus to the cytoplasm, it did not serve any larger

purpose. And the long stretches of DNA that did not contain information

for building proteins were unimportant.

In 1993, scientists started to find the first

hints that this long-standing model of how

life works might not explain everything.

Researchers studying Caenorhabditis elegans

worms started finding evidence that short

18–25 nucleotide-long snippets of RNA 

produced from genes that did not encode

protein might be bigger players in the workings of cells than had been realized. These

snippets started turning up in a number 

of different species, showing remarkable

conservation and suggesting that some 

fundamental piece of the cell was starting 

to make itself known (Figure 1). 

Fast forward to the present day. The

importance of these short RNA pieces—now

dubbed microRNAs—as epigenetic regulators of cell development, survival, and 

disease is becoming ever clearer. And their

popularity as a research topic has exploded.

“The basic science of microRNAs is just fascinating,” said Curtis Harris, M.D., Chief of

CCR’s Laboratory of Human Carcinogenesis,

who studies microRNAs as prognostic tools

in cancer. “How the microRNA is processed

from its gene, its function in normal cell 

biology, in development, and in disease—

since microRNAs are relatively newly 

discovered, all of these features are still

being worked out.” A search of the PubMed

database turned up 2,611 papers published

on microRNAs since 2001, with 1,027 published in 2007 alone.

The links between microRNAs and

cancer are also now well appreciated. “The

cancer field has now become very excited by

microRNAs, in part because they are so

new,” noted Harris. Cancer researchers also

have a clearer view of microRNAs as actors

in carcinogenesis. Based on this knowledge,

researchers like Harris are investigating 

how to turn our growing knowledge of

microRNAs into clinical tools for cancer

prognosis and therapy. 

Shooting the Messenger

MicroRNAs are like transcription factors for

RNA. Just as transcription factors control a

gene’s transcription into messenger RNA,

microRNAs control a messenger RNA’s

translation into protein. But instead of 

promoting gene expression, as transcription

factors do, microRNAs impede it:

MicroRNA-bound messenger RNAs do not

get translated, effectively silencing the 

gene from which they were transcribed.

“MicroRNAs bind to the messages of proteincoding genes, either changing the stability 

of that message or the translation of that

message into protein,” explained Harris. 

The number of microRNA genes

tucked away in the genome is unclear, but by

some estimates there may be as many 

as a thousand. Researchers estimate 

conservatively that about one-third of all

One Man’s Junk DNA Is Another

Man’s Treasure: MicroRNAs and the

Next Big Thing in Cancer Prognosis

feature

A search of the PubMed 

database turned up 

2,611 papers published on

microRNAs since 2001, with

1,027 published in 2007 alone.
ccr connections | v OLUME 2, N O . 1 | 2008 1 7

protein-coding genes may be controlled to

some extent by microRNAs. They can have

such widespread effects because they tend to

act globally. “Because they are short and their

‘seed’ sequences [the first six nucleotides in a

microRNA, which act as binding sites] are

somewhat degenerate, they physically interact with messages that they don’t exactly

match,” Harris noted. “So a single microRNA

may target 10, 50, maybe even a 100 messages in different genes or pathways.” 

Drawing the Lines

The first paper suggesting a link between

microRNAs and cancer, published in 2002 by

a colleague of Harris, The Ohio State

University’s Carlo Croce, M.D., reported that

a genomic region deleted in about half of all

cases of B-cell chronic lymphocytic leukemia

(B-CLL) housed genes for two microRNAs.

The development of techniques for microarray and bead-based flow cytometry analyses

of microRNA expression soon led to the discoveries of microRNA signatures unique to

specific tumors and their cellular origins. 

As the research on microRNAs 

and cancer has gone deeper, particular

microRNAs have begun to stand out as

potentially causative agents. For instance,

microRNAs called miR-155 and the miR-17-92

cluster act like oncogenes, while miR-15a

and miR-16-1 appear to function as tumor

suppressors. The genetic lesion Croce identified in B-CLL included the genes encoding

miR-15a and miR-16-1. 

“We are finding that microRNAs can

serve a range of purposes in the context of

cancer and cancer treatment,” said Harris.

“They can tell us a lot about the basic biology

of cancer and about what pathways are

involved and what might be good targets for

therapeutic development. One can, at least

in preclinical studies, knock down the

expression of a specific microRNA with an

antisense strategy and see an anti-tumor

effect. They can also be good clinical biomarkers, useful tools for diagnosis and,

maybe, for predicting therapeutic outcome.”

Translating Science Together

It was the role of microRNAs as developmental players in cancer, combined with

Croce’s B-CLL paper, that gave Harris his

entrée into the world of microRNAs. “When

Carlo made what I think was a seminal

observation that microRNAs were associated

with cancer, that seemed to be a very exciting

finding and one that I thought might have

relevance to solid tumors.” 

Having known Croce for some time,

Harris contacted him and suggested that

they work together to look at microRNA 

profiles in solid tumors. Joining forces, their

laboratories produced, in early 2006, an

examination of the microRNAomes (the

total palette of microRNA expression within

a cell) of tissues from a spectrum of solid

tumors (e.g., lung, breast, stomach, prostate,

and colon). “This paper was one of the first

to indicate the extensive involvement of

microRNAs in the pathogenesis of solid

tumors,” reported Harris. It also suggested

that microRNA expression could influence 

cancer development by controlling proteincoding oncogenes and tumor suppressors

(Figure 2).

Peeking into a Tumor’s Future

Apart from the questions of development

and pathogenesis are those of progression.

The ability to predict an individual’s clinical 

outcome or risk for recurrence has been an

(I m a g e: J . Kel l y )

feature

Figure 1: The roles of microRNAs—simple short pieces of RNA that do not encode

protein—in the control of nearly all critical cellular processes have gained widespread and rapid appreciation.
“MicroRNAs, in this context, are a

new class of biomarkers that will be

useful not only with clinical stage,

but also with other biomarkers such

as genomic or proteomic changes.”

1 8 ccr connections | v OLUME 2, N O . 1 | 2008

enigmatic target for researchers but one that

could have great benefit. Could microRNAs

give clinicians insight into how a tumor will

behave as time goes on? 

Harris framed the prognosis problem

in the context of lung cancer. “Frequently a

surgeon operating on a patient with stage 1

lung cancer will tell them after surgery, ‘We

got it at an early stage; hopefully we got it

all.’ In fact, about half of these patients will

have recurrence with distant metastases

over a five-year period. If surgery isn’t sufficient, we bring in our armamentarium of

adjuvant therapies, but because of the side

effects, you don’t want to use them unless

you think there is a good reason to.

“If you knew,” Harris continued,

“which of these patients will have a good

prognosis, and perhaps need less therapy

and screening, versus those who will have a

poor prognosis and need more therapy and

need to be screened more frequently, you

would have greater justification to offer 

particular adjuvant therapies to a particular

patient. This need for better prognosis 

has fueled the whole biomarkers field.

MicroRNAs, in this context, are a new class

of biomarkers that will be informative not

only when combined with clinical stage, 

but also with other biomarkers such as

genomic or proteomic changes.”

With this reasoning in mind, Harris,

Croce, and their colleagues started narrowing their view of microRNA expression 

profiles to focus on prognosis. In 2006, they

released a paper, authored by now-Harris

lab alumnus Nozomu Yanaihara, M.D.,

Ph.D., revealing that the expression pattern

of microRNAs in lung tumors correlated not

only with tumor type, but also with prognosis.

It was one of the first studies to tie microRNAs

to a patient’s prognosis following surgery,

independent of tumor stage.

Broad Effects

“The same things we know about stage 1

lung cancer and the limitations of clinical

staging can be said for stage 2 colon cancer,”

Harris noted. “There is a similar need to

identify those individuals who have a good

or poor prognosis independent of stage. You

feature

can get a good idea on a population level

who is going to face recurrence and who is

not, but on an individual basis, you have to

be cautious when deciding who needs what

therapy and how aggressively a patient

needs to be treated.”

Knowing that they could predict lung

cancer outcomes, Harris and Croce teamed

up again to see if they could achieve the

same results in colon cancer. The time and

place for this study were ideal—Harris has

studied colon cancer for a number of years

and has a long-standing cohort of patients

from whom he has collected tissue and

detailed clinical and family histories. “In

addition,” Harris said, “I knew of a cohort in

Hong Kong that would provide us with a validation population that would allow us to

confirm any results we found in our locallybased cohort. I wanted to make a diverse

comparison by looking at two very different

cohorts. If a typical U.S. population and, in

this case, a typical Chinese population show

the same result, it is more likely that your

data will be generalizable to a broad range of

people with colon cancer.”

And once again, Harris’ and Croce’s

collaboration has proven fruitful. A new

paper, released in January of this year and

co-authored with Yanaihara by Harris laboratory Cancer Prevention Fellow Aaron

Schetter, Ph.D., M.P.H., and Postdoctoral

Fellow Jane Sohn, Ph.D., showed that

microRNA profiles could be used to predict

both prognosis and clinical outcome,

another first in the microRNA world. In

addition, they found that the levels of 

(Photo: C. Harris, CCR)

Figure 2: The overabundance of microRNAs like miR-21 in tumor tissue (right,

adjacent to normal tissue expressing low levels of miR-21) may represent both 

a causative factor in colon and other cancers and an opportunity for the 

development of new prognostic tests and therapeutic agents.
ccr connections | v OLUME 2, N O . 1 | 2008 1 9

The question of predicting survival and

response is not limited to colon and lung

cancers. One cancer for which there is a significant need for new prognostic tools is liver

cancer. Currently, surgical resection or transplantation are the best options available for

liver cancer patients; however, based on

assessments of liver function, tumor size,

and stage, only 10 to 20 percent of patients

are eligible for these surgical options. Even

those who are able to undergo surgery face

an uncertain future; the frequency of metastasis and/or recurrence is very high.

(Photo: C. Harris, CCR)

feature

Figure 3: Comparisons of miR-21 abundance in colon cancer tissues with patient 

outcomes reveal a striking correlation—the more of this microRNA that is present in

patients’ tumor cells, the worse their prognosis. The Harris lab conducted the first

study of this link between colon cancer survival and microRNA expression.

a specific microRNA called miR-21 also 

correlated with cancer stage: The later the

stage, the more miR-21 they found in 

the tumor cells. MiR-21 is one of the

microRNAs that have, over time, made an

appearance in other cancers as well. Thus,

there is a growing body of evidence to 

suggest that it might play a fundamental

role in the progression of colon and other

cancers (Figure 3). 

A New View of Liver Cancer

Which means miR-21 may also fit 

the bill as a therapeutic target. “While 

surgery is the first-line treatment of colon

cancer,” Harris noted, “if there is evidence

of metastases, one still gets the best

response overall by using fluorouracilbased therapies, which are nearly 50 years

old. We need novel options for colon 

cancer patients, and maybe targeted

microRNAs could be a good one.” 

Knowing that microRNAs were starting

to show promise for prognosis in other

tumors, Xin Wei Wang, Ph.D., Head of the

Liver Carcinogenesis Section in CCR’s

Laboratory of Human Carcinogenesis, began

to look at whether such patterns could be

applied to liver cancer as well. By comparing

cancerous and noncancerous liver tissues,

Wang and his collaborators identified 20

microRNAs whose expression correlated

with risk of metastasis and did so with

greater accuracy than classical pathology

staging. They also found that this pattern

itself could be used as an independent

measure for predicting a patient’s clinical

outcome. 

Wang looks at these results in terms 

of clinical benefit. Methods for microRNA 

isolation and analysis are advancing 

rapidly, which creates conditions for turning

microRNA profiling into a standard 

procedure for liver cancer patients. Having 

a profile in hand that can distinguish highand low-risk patients would allow clinicians

to decide early after a diagnosis how aggressive a treatment approach to take and give

insights into how to personalize treatment 

for an individual patient.

Looking to a MicroRNA-Based

Future

“We are only five or so years separated in

time from the first suggestions that

microRNAs could be involved in cancer,” 

said Harris. “In that short time, we have come

very far, and in my opinion, microRNAs are

going to be very significant biomarkers for

diagnosis and prognosis in a number of 

cancers. I also anticipate that microRNAs

may be useful clinical targets, which is a

longterm goal for us to determine.” 

But he is the first one to declare that

there is a great deal more work to be done.

“I’d like to see our prognosis results 

replicated in a number of different populations, so that we can see how broad they

are,” mused Harris. “Ours was the first report

on the use of microRNAs to predict therapeutic outcome, and as such these data

need to be confirmed. But it is an exciting

time and an exciting opportunity, being

engaged at the early stages of translating a

fundamental discovery.”
2 0 ccr connections | v OLUME 2, N O . 1 | 2008

feature

Curtis C. Harris, M.D. 

Chief, Laboratory of Human Carcinogenesis 

“It all started in a small farming community in Kansas and at

state science fairs,” Curt Harris quipped when asked about his

background. But he came to CCR with an inborn instinct for

translational medicine. “One of my medical school mentors, with

whom I had done some collaborative research, suggested that I

should continue my work at NCI. I started out with a small laboratory while finishing my clinical training and haven’t ever

thought about leaving.” 

His research interests, at first glance, cover a range of 

topics. “My lab’s research is diverse, which reflects being a physician-scientist. We have a strong motivation for understanding

basic research and translating that knowledge into the clinic.”

What truly excites him, though, are moments of unexpected 

convergence. “I love it when there are two parallel lines of research

in the laboratory, and there is a connection that we never would

have predicted, leading us into something much more interesting.” 

This eye for convergence has fueled his work on biomarkers

and prediction. “We have done a lot of work on molecular pathogenesis of cancer and how normal cells become cancer cells,”

Harris said. “We try to look at cancer from a scientific standpoint,

a clinical standpoint, and a public health standpoint.”

Harris finds working with his growing web of laboratory 

alumni a fruitful and enjoyable aspect of his job. “We maintain what

we like to call the ‘LHC Family.’ I find it very satisfying to collaborate with former fellows who have cultivated their own independent

careers and with whom I can have a collegial relationship.”

Harris earned his medical degree from the University of

Kansas School of Medicine and did his clinical training at the

University of California at Los Angeles and at NIH.

A Diverse Team 

(Photo: R. Baer)

(Photo: Courtesy of N. Yanaihara)

Nozomu Yanaihara, M.D., Ph.D. 

Harris Laboratory Alumnus

Nozomu Yanaihara worked with the Harris lab from 2004 to 2007 as

a Research Resident and a Visiting Fellow from the Jikei University

School of Medicine in Tokyo, Japan. “I am a gynecologist and an

obstetrician by training, with a focus on gynecological oncology.

When I decided to come to the National Cancer Institute for additional experience, one of my mentors, a former Fellow in Curt’s

lab himself, suggested I contact him.”

Now an Assistant Professor of Obstetrics and Gynecology

back at Jikei University, Yanaihara maintains an active relationship with his colleagues in the Harris laboratory. “One of the

biggest things I learned at CCR,” Yanaihara noted, “was that while

researchers from different countries may struggle with language

barriers, there are no barriers in research as long as we have

shared goals.”

His interests in microRNA and cancer, fueled by his work at

CCR, have now crossed into his work in Japan. “I took part in several microRNA-related projects while at NCI, including the lung

cancer prognosis prediction project. I am now applying the techniques and results that I brought back to prognostic research in

gynecological cancers. My hope is to carry out this work collaboratively with Curt and others back in the United States.”

Yanaihara received both his M.D. and his Ph.D. from Jikei

University.
ccr connections | v OLUME 2, N O . 1 | 2008 2 1

feature

(Photo: R. Baer)

Aaron Schetter, Ph.D., M.P.H. 

Cancer Prevention Postdoctoral Fellow

Aaron Schetter’s path to the Harris lab has followed a route that

places him squarely at the intersection of public health and basic

science. “I started off doing basic research studying cell biology in

C. elegans,” said Schetter. “After I finished my Ph.D., I wanted to

switch to a field that was more relevant to human disease. After

deciding to study cancer, I joined the NCI’s Cancer Prevention

Fellowship Program.”

Launched in 1987, this program trains multidisciplinary

experts in cancer prevention. Scientists, clinicians, and other

health professionals are encouraged to earn an M.P.H., followed

by mentored research with NCI investigators. “It’s given me a

completely different set of skills than what I developed as a Ph.D.

student,” Schetter noted.

After completing the academic portion of the program,

Schetter looked for a laboratory where he could put those skills to

work. “It’s difficult to find labs where you can do both basic science

and epidemiology well. Curt’s lab is a large and diverse group, with

people doing basic science and ones who exclusively do this kind

of epidemiologic work, so to me it seemed a good fit.”

The translational aspect of Harris’ microRNA research 

also drew him in. “I saw how the microRNA work could rapidly

turn into something that could affect human disease itself.

MicroRNAs are pretty easy to work with in this regard. You can see

which ones are altered in colon cancer and then move quickly

into functional studies testing your hypotheses.”

Schetter’s experiences give him some pretty broad options

for the future. “I haven’t decided yet whether to go the academic

route or the industry route, but regardless I hope to continue in

the field of discovering and testing biomarkers and therapeutic

targets in cancer.”

Schetter earned his Ph.D. at Cornell University and his

M.P.H. at the University of California at Berkeley.

Jane Sohn, Ph.D. 

Postdoctoral Fellow 

With a research background in microbiology, Jane Sohn brings a

different perspective to research on cancer. “I came to Curt’s lab

to work on colon cancer and a related disease called inflammatory

bowel disease (IBD). It’s thought that bacteria contribute to 

cancer risk by inducing inflammation. Though the etiology of IBD

is not known, it is a disease of inflammation, and IBD patients

have an increased risk for colon cancer.”

Her current work stems from a realization she had while

working on her Ph.D. “I was working in a laboratory that focused

on bacterial pathogenesis and realized that I was interested in

looking at the interface between inflammation and cancer.

MicroRNAs may help us better understand this interface.” 

Sohn finds the environment within the Harris lab to be truly

unique. “Curt’s lab does three different things: basic research,

translational research, and molecular epidemiology. NIH encourages basic scientists to collaborate with others doing translational

science or epidemiology, and here it is already happening just

within this lab.

“I came from a basic science setting,” Sohn noted, “and now

have an appreciation for human disease that I didn’t have before.” 

Sohn did her doctoral work at the Massachusetts Institute

of Technology.

Aaron Schetter, Ph.D., M.P.H. (left), and Jane Sohn, Ph.D.

(right)
2 2 ccr connections | v OLUME 2, N O . 1 | 2008

I clinical trial in patients with CD25-expressing

hematological malignancies that had failed

previous treatment. 

LMB-2 was well tolerated by the

study’s patients. More importantly, several

patients on the trial experienced a disease

response within one week of treatment with

LMB-2. In particular, four trial participants

with a rare cancer called hairy cell leukemia

(HCL) all demonstrated major responses to

the immunotoxin. In one patient, the count of

malignant cells dropped to an undetectable

level. The LMB-2 trial clearly illustrated that

powerful, targeted cell-killing agents could 

be designed and produced using genetic

engineering. 

Though the results were encouraging,

Pastan and his colleagues wanted to develop immunotoxins to treat more common

cancers (only 900 to 1,000 people are diagnosed with HCL in the United States each

year). They decided to target CD22, a membrane protein commonly expressed on Bcell lymphomas and leukemias. While CD22

is also expressed on normal B cells, it is not

present on the stem cells that generate B

cells, allowing the body to readily regenerate

its B cells and making CD22 a potentially

good target for immunotoxin therapy.

Pastan teamed up with David J.

FitzGerald, Ph.D., Head of the LMB

Biotherapy Section, and Kreitman to design

a new immunotoxin—dubbed BL22—

to deliver PE to CD22-expressing cells and

test it in a Phase I clinical trial in patients

with chemotherapy-resistant non-Hodgkin’s

lymphoma (NHL), chronic lymphocytic

leukemia (CLL), and HCL. 

Immunotoxins are proteins designed to

deliver a lethal blow directly to cancer cells.

Each has two parts: a custom-designed antibody that can home in on a specific target

on the surface of the cancer cells and a

toxin, which delivers the fatal blow. When

the antibody binds to its target, the whole

complex is pulled inside of the cancer cell,

where the toxin can do its work. 

The team of CCR researchers, led by

Ira Pastan, M.D., the Laboratory’s Chief,

shares a drive to do more for people with

cancer. They have already begun to see their

determination to devise better immunotoxins pay off, but they are continuing to dig

deeply into the workings of the cell to learn

why immunotoxins work in some patients

and not in others. And their ability to quickly

translate these insights from the labs at CCR

to the infusion rooms at the NIH Clinical

Center is helping them move deftly toward

new anti-cancer therapies. 

After spending most of his career

studying cell surface receptors and their signaling pathways, Pastan shifted gears.

“Since I was a physician trained to do

research,” he recalled, “I wanted to use what

I knew to do something relevant to the treatment of cancer.” He began working with

Pseudomonas exotoxin A (PE), a bacterial protein with potent cell-killing activity, and

became intent on finding a way to deliver

the lethal toxin to tumor cells. 

Proof of Concept

When Pastan first started working with

immunotoxins in the early 1980s, existing

PE-based immunotoxins had several limitations. They were complex and costly to 

produce, and their large size prevented them

from efficiently penetrating bulky tumors. 

Pastan was able to make several

important improvements by using recombinant DNA techniques to engineer smaller,

more nimble immunotoxins that could be

produced inexpensively in large quantities.

“That conversion,” said Pastan, “from coupling toxins to antibodies using chemistry

to using genetic engineering to make these

molecules, was really a big breakthrough.”

His team’s first target was a receptor,

CD25, found on the surface of many B- and

T-cell cancers as well as some normal B and

T cells. Using an immunotoxin called LMB2, Pastan and CCR colleague Robert J.

Kreitman, M.D., Head of the LMB Clinical

Immunotherapy Section, conducted a Phase

Just as computer makers continue to devise smaller, faster, and more powerful

laptops, the scientists in CCR’s Laboratory of Molecular Biology (LMB) will

not stop re-engineering their cancer-killing immunotoxins until they have 

created the optimum life-saving treatment. 

A Better 

Immunotoxin

feature

Through inspired engineering, proteins that enter and kill

cancer cells are finding their place among cancer treatments. 

The LMB-2 trial clearly illustrated that powerful, targeted

cell-killing agents could be

designed and produced using

genetic engineering. 

Ira Pastan, M.D.

(Photo: R. Baer)
ccr connections | v OLUME 2, N O . 1 | 2008 2 3

Even at low doses, many HCL patients

responded to BL22. With increased doses,

their responses grew more frequent and

more dramatic. The cancer cells of many

HCL patients completely disappeared from

the blood, bone marrow, and spleen, while

normal blood cells—which are commonly at

dangerously low levels in patients with

advanced blood malignancies—returned to

normal levels. 

More than half of the patients with

advanced HCL were able to return to a normal life, and many remain in complete

remission more than three years after the

initiation of treatment. In a 2005 review 

article, Pastan noted that “This clinical

experiment, in which more than half of the

patients with an advanced malignancy were

able to resume a normal life, was certainly

the most rewarding experiment in my

research career.”

Beyond Hairy Cell

Although the responses in HCL patients to

BL22 were profound, results in patients

with other cancers were less encouraging.

This information left the Pastan research

team with a challenging decision. Should

they continue to work on BL22 and try

to get it approved for treatment of the 

relatively rare HCL? Or should they try to

engineer an immunotoxin that could effectively treat more common CD22-expressing

cancers like CLL, which afflicts more than

15,000 each year? 

The group decided they could make

an immunotoxin that did both. They knew

that they had to find a way to deliver more

immunotoxin to CLL cells. “CLL cells don’t

have as much CD22 as HCL cells,” Kreitman

explained. Indeed, a single HCL cell might

have as many as 70,000 CD22 proteins on its

surface, giving BL22 ample opportunity to

latch onto the cell. CLL cells often have

fewer than 1,000. 

They needed an antibody that binds

with higher affinity (more tightly) to CD22.

This would make it less likely that the

immunotoxin will fall off once it binds to the

cell, increasing the probability that it will be

ushered inside. 

To optimize BL22’s affinity, the Pastan

lab utilized a method called hot spot 

feature

While hematological 

malignancies are well suited

for immunotoxin treatment

because they are easily

accessible via the 

bloodstream, solid tumors 

are harder to reach and 

to penetrate. 

mutagenesis, which mimics the way the

immune system would do it. They altered

specific regions of their anti-CD22 antibody

and looked for mutants that bound more

tightly to CD22. When they found what they

were looking for, they built HA22—which is

up to 20-fold more powerful than its predecessor. Pastan and his colleagues recently

launched a Phase I/II trial to find out if HA22

can more effectively combat both cancers.

Initial results have been promising, and the

team hopes to know within the next few

years whether the new immunotoxin will

prove to be an improved treatment against

CD22-expressing malignancies. 

Raffit Hassan, M.D.

David J. FitzGerald, Ph.D.

(Photo: M. Spencer) (Photo: M. Spencer) (Photo: S. Neal)

Robert J. Kreitman, M.D.

Tackling Solid Tumors 

While hematological malignancies are well

suited for immunotoxin treatment because

they are easily accessible via the bloodstream, solid tumors are harder to reach and

to penetrate. Pastan and his group, however,

believe that these barriers are surmountable

and have set out to identify a target to help

them confirm their belief. 

More than ten years ago, Pastan and

his LMB colleague Mark Willingham, M.D.,

now at Wake Forest University School of

Medicine, identified mesothelin when they

were searching for proteins expressed in

ovarian cancer cells. Mesothelin is highly

expressed in more than 70 percent of ovarian cancers, as well as a high percentage of

mesotheliomas (cancers of the membrane

that encapsulates most of the major

organs), lung adenocarcinomas, and gastric

and pancreatic cancers. 

Though its function in cancer cells has

not been well characterized, there is some

evidence that mesothelin’s interactions with

another protein, CA-125, may promote the

spread of ovarian cancer. Mesothelin is normally expressed on the epithelial cells that

line body cavities but not in essential organs

such as the heart, brain, liver, and kidneys.

“Since there is high expression in tumors,

but very little expression in normal tissue, it

makes a good target for antibody-based

therapy,” said Raffit Hassan, M.D., Chief of

LMB’s Solid Tumor Immunotherapy Section. 
2 4 ccr connections | v OLUME 2, N O . 1 | 2008

To target mesothelin, the Pastan lab 

engineered an immunotoxin called SS1P. In

laboratory tests, SS1P had impressive activity against cells from ovarian cancer

patients. It also made tumors shrink and

prevented lung metastases in mouse models. Based on these preclinical data, Pastan,

Hassan, and Kreitman launched two separate Phase I clinical trials to test SS1P in

patients with recurrent mesothelioma, ovarian cancer, and pancreatic cancer. 

SS1P did not cause toxicities in any

essential tissues, such as heart and liver, but 

it did cause an uncomfortable but not lifethreatening inflammation of the body cavity

lining. Patient responses to treatment were

modest, but they were considered encouraging when compared with results seen with

other immunotoxins and antibody-based

therapies used against solid tumors. 

There currently are limitations to SS1P’s

effectiveness. Like other immunotoxins and

antibody-based therapies, SS1P has trouble

penetrating solid tumors. Such tumors 

have poor vascular and lymphatic systems,

impeding the delivery of treatment agents.

They also have high internal pressures, which

favor an outward rather than an inward flow 

of material. In addition, the Pastan lab has

found that solid tumors shed large amounts 

of mesothelin from their cell surfaces, which

may absorb the immunotoxin and keep it 

from interacting with and killing target cells. 

feature

Interestingly, Pastan’s group has

found that tumor-bearing mice treated first

with a chemotherapy agent called Taxol® and

then with SS1P show dramatic tumor regressions due to a synergistic interaction of the

two drugs. “We think the synergy is caused

by two things,” Pastan explained. “We think

the Taxol kills some tumor cells and disrupts

the organization of the tumor, allowing the

immunotoxins to penetrate better. And the

shed mesothelin, which is very high in the

tumor, falls to very low levels and doesn’t

soak up the immunotoxin. Taken together,

this strategy gives us fantastic synergy.”

Pastan and Hassan are preparing to

test their theory in a Phase II clinical trial

combining chemotherapy with SS1P. The

researchers hope that this combination

approach will be a newly effective strategy for

targeting solid tumors with immunotoxins. 

The other protection solid tumors

have that hematologic malignancies do not

is a strong immune system on the part of the

To be effective against solid tumors like those of ovarian cancer, immunotoxins like SS1P (which targets a protein called

mesothelin) need to be able to penetrate deeply into the tumor. In mice, treating tumors with a chemotherapy agent like Taxol®

before administering SS1P greatly increases the immunotoxin’s activity, possibly in part by disrupting the tumor’s structure.

(Image: J. Kelly)

patient. “So far we can only give patients

with solid tumors one or two cycles before

their immune systems mount a very good

antibody response to the toxin and neutralize

it,” Pastan lamented. This issue is rarely a

problem in patients with hematological

malignancies because their immunity is

much weaker, having been suppressed by

both the disease and previous chemotherapies. As a result, these patients can receive

multiple cycles of therapy. 

Pastan’s team is pursuing several

strategies to enable the immunotoxins to

evade the immune system. For example,

they are trying to identify regions of the toxin

recognized by patients’ immune systems

and then modify or delete them using protein engineering.

Immunotoxins of the Future

The CCR research team continues to learn

how to make immunotoxins even more

deadly to cancer cells and to identify new

therapeutic targets. Scientists know that

the PE toxin inhibits protein synthesis once

inside the cell but not how this triggers cell

death. FitzGerald is exploring where the

toxin goes after gaining entry; perhaps, he

said, “Different cell surface targets may shuttle the toxin along different intracellular

paths. If one pathway offers greater benefit

to patients, perhaps an immunotoxin could

be engineered to follow that better path.” 

Even with cutting-edge

high-throughput 

techniques, finding the

ideal immunotoxin target

is a difficult task. 
ccr connections | v OLUME 2, N O . 1 | 2008 2 5

From Dangerous Fatigue to

an Energetic Retirement

feature

As he neared his fiftieth

birthday, Dave Brenneman,

an engineer for the State 

of Pennsylvania, began to

notice that his energy was

flagging. He tried to convince himself that it was all

in his head or the inevitable

result of getting older, but

when his fatigue persisted,

he decided to get a checkup. His family doctor told

him that his blood counts were lower than normal. A visit to a local oncologist and a series

of additional tests revealed the “odd-looking” cells of hairy cell leukemia (HCL) in his blood. 

Like many HCL patients, Brenneman was treated with the drug cladribine. His blood

counts climbed back to normal over the next few months. “I was feeling great,” Brenneman

related, “but after approximately three years, my counts started to drop again.” Two years

later, he received another round of cladribine, but this time the cancer began to come back

after only a year.

A proactive patient, Brenneman turned to the Internet to find other potential treatment

options for HCL. He stumbled across information on a clinical trial being conducted on a

new drug called BL22 and contacted Robert Kreitman, M.D., at CCR to find out more. 

Kreitman and his colleagues looked carefully at Brenneman’s cancer and decided that he was

a good candidate to receive BL22. “His bone marrow was 70 to 80 percent HCL cells,”

Kreitman recalled, “and his neutrophils, which help fight infection, had decreased to a count

of 508. Less than 500 is considered life-threatening.”

Brenneman had reservations about joining a clinical trial. “I was very skeptical, but as I

learned more and talked with Kreitman and his staff, I became more comfortable with the

idea.” He traveled to the NIH Clinical Center to receive his first cycle of the immunotoxin in

September 2005. That one cycle was enough to trigger a complete remission. “His neutrophil

count four weeks later was normal and the hairy cells were gone from his bloodstream,”

Kreitman explained. “Repeat tests showed no hairy cells left in his bone marrow either.” The

benefits of that single cycle of BL22 have lasted; Brenneman’s bone marrow biopsies and

blood tests have remained clear for over two years. 

Thanks to BL22 and the dedicated team of CCR researchers, Dave Brenneman is making the most of his recent retirement. He has plenty of energy to maintain the dairy farm that

has been in his family for three generations, work on his classic car, and spend time with his

wife, children, and grandchildren. His strong response also illustrates the potential of BL22

and next-generation immunotoxins for treating HCL and—with continued improvements—

other cancers as well. 

(Photo: Courtesy of D. Brenneman)

Even with cutting-edge high-throughput techniques, finding the ideal immunotoxin target is a difficult task. Proteins must

meet several stringent criteria to qualify 

as potential immunotoxin targets. They

must appear on the cell surface, because

immunotoxins are unable to cross cell 

membranes unassisted. And they must be

expressed mainly on cancer cells and

nonessential normal cells, not in essential

tissues such as the kidneys, heart, and brain;

any attack by an immunotoxin on these 

tissues could result in serious side effects. 

Pastan and his colleagues found one

new receptor by scouring the literature to

identify proteins that are selectively

expressed in certain types of cancers.

FCRL1 is part of the Fc receptor-like (FCRL)

family of proteins, which is expressed by

different cells of the immune system.

FCRL1 is selectively expressed on B cells

and is present at high levels on the tumor

cells of CLL patients. The scientists have

generated two immunotoxins that recognize

FCRL1, both of which have shown promise

in the culture dish. These new immunotoxins are now ready for testing in animals.

The Pastan lab is also particularly

interested in identifying new targets for

breast and prostate cancers, since people

can survive without the normal tissues

associated with these organs. Searching

through gene databases, Pastan lab Staff

Scientist Tapan Bera, Ph.D., and colleagues

in the lab have found a potential target for

prostate cancer, a protein called NGEP, that

is expressed in prostate cancer and normal

prostate cells but not in essential tissues.

The team is now developing antibodies to

NGEP, which will be used as the basis for

new immunotoxins that they hope will represent a new treatment option for prostate

cancer patients.

Pastan’s work has been over 20 years

in the making, but his progress in turning

his basic discoveries into effective tools has

been accelerating quickly. Bera joined the

Pastan lab in 1995, “to be involved in a

research project where you can see the clinical benefit right away.” He has not been

disappointed.
2 6 ccr connections | v OLUME 2, N O . 1 | 2008

feature

(Photos: C.R. Lee, CCR)

When Stuart H. Yuspa, M.D., began 

studying the skin in the late 1960s, scientists

understood some of the basic biology of skin

cancer. They knew what was required to 

produce and diagnose benign and malignant

tumors—but not much more. “We’ve made

remarkable leaps since then,” says the 

Co-Chief of CCR’s Laboratory of Cancer

Biology and Genetics (LCBG). 

No

Longer

Skimming

the

Surface 

of 

Skin

Cancer 

Squamous cell carcinoma (top) is a relatively aggressive skin cancer,

and it shares many of the features of lethal solid tumors of the internal

organs. The Yuspa laboratory’s efforts to model the biology of skin

have given great insight into the genesis and treatment of this and

other major skin malignancies, such as basal cell carcinoma 

(middle), and malignant melanoma (bottom).

Stuart H. Yuspa, M.D.

( P h oto: E. Br a n so n )
learn how to culture the skin,” according to

Molly Kulesz-Martin, Ph.D., who was a

Postdoctoral Fellow in Yuspa’s lab from 1979

to 1981 and is now Director of Research and

Professor in the Department of Dermatology at

Oregon Health & Science University (OHSU).

When Kulesz-Martin joined the Yuspa

lab, very few people were working on epithelial cells, even though most human cancers

arise from them. “Everybody was working on

cells that were easy to grow, but there was

no way to assure they were like some particular organ in the body. And they weren’t,”

she recalled. 

“Stu was working out ways to grow

epithelial cells so they behaved as they do in

the body. We had to find a way to make culture conditions good for growing epithelial

cells,” said Kulesz-Martin. Her work built on

earlier laboratory observations that reducing

calcium levels enabled cells to grow for

much longer than two weeks. From that, she

created a transformation assay to quantify

the strength of various carcinogens.

Kulesz-Martin’s experiences in Yuspa’s

lab set the path for her future as a

researcher. At OHSU, she continues to work

with mouse and human cells to determine

the initial changes and later insults that

push a cell from normal to malignant to

metastatic. “I didn’t fall far from the tree,”

she admitted. “I still want to study cancer,

still study skin as a model, and I’m still

ccr connections | v OLUME 2, N O . 1 | 2008 2 7

feature “Now we know the genetic changes associated with each stage of cancer development

and how those genetic changes translate to

biochemistry in each stage,” Yuspa, a specialist in squamous cell skin cancers,

explained. “And we have markers to recognize where we are in the progression from

normal to malignant.” 

Yuspa added, “We also know a lot

more about normal skin. We know how skin

homeostasis is controlled and the pathways

that regulate it. That’s extremely important

because you have to understand normal to

understand abnormal.” To generate this

understanding, over the last 36 years the

Yuspa laboratory has developed in vitro 

models that recapitulate the normal growth

and differentiation of skin epithelial cells

called keratinocytes—precursors to the

squamous cells that give squamous cell skin

cancer its name. The models also reproduce

each stage of carcinogenesis as it occurs in

mouse skin cells.

These model systems have been “really

important for understanding mechanisms of

cancer,” said Adam Glick, Ph.D., a former

Postdoctoral Fellow and Principal Investigator

who still collaborates with Yuspa. 

And the work is moving from discovery

to application. Yuspa, Glick, and their colleagues recently identified genetic markers

that distinguish low-risk benign skin tumors

from high-risk tumors in mice and opened

possibilities for targeted therapies that

block early tumors from progressing to invasive lesions.

Yuspa’s models are making inroads for

other cancers as well, since similar epithelial

cells are involved in cancers of many internal

organs, such as the lungs, head and neck,

esophagus, colon, and stomach.

A Good Decision

Yuspa first came to NCI in 1967 to work in

the lab of Richard R. Bates, Ph.D., who was

working on skin carcinogenesis. “There were

two of us and a few technicians,” Yuspa

recalled. “We did some very nice work looking at carcinogen binding to DNA and how

that caused mutations. I loved it! I stayed for

the two years of my United States Public

Health Service Commissioned Corps obligation, then asked to stay a third.” The young

M.D. then left to pursue his clinical training

to see what aspect of medicine he liked

most—research or clinical work. 

“Dick said he’d hold the spot for me,

which would be impossible today, but it was

the early days of the War on Cancer,” Yuspa

continued. After finishing his medical residency training—and realizing that the lab was

where he wanted to be—Yuspa returned to

the Bates lab to work on the skin model. He

became a Senior Investigator at NCI in 1972.

The Place to Be 

“Stu’s lab was a place that people came to

from all over the world—and still do—to

Each type of skin cancer—melanoma, basal cell

carcinoma, and squamous cell carcinoma—

arises in different cells within the skin.

Melanoma forms in melanocytes (skin cells that

make pigment) and is the most dangerous form

of skin cancer. Basal cell carcinoma forms in cells

found in the hair follicles. Basal cell cancers are

the most frequent human tumor in Caucasians,

but they grow slowly and rarely spread to other

parts of the body. 

What Kind of Skin Cancer?

Squamous cell carcinoma, the focus of the work

of Stuart Yuspa, M.D., begins in squamous cells

(flat cells that arise from keratinocytes and form

the surface epidermis of the skin). They are not

the most frequent skin cancers, but they share

many characteristics—genetically and biochemically—with highly lethal cancers that arise

in internal organs like the lung, head and neck,

esophagus, and stomach. 

(Gr a p h ic: N CI)

The skin’s epidermis harbors the

keratinocytes and melanocytes

that give rise to skin cancer. The

Yuspa laboratory’s efforts to model

the biology of skin have given

great insight into the genesis and

treatment of skin cancers.
Colleagues and former students of Stuart

Yuspa, M.D., unanimously point out his

generosity with his ideas and time—and his

love of science. 

He has built a dynamic community of

researchers exploring skin cancer. He sees

mentoring as a happy obligation. “The only

legacy that will be remembered is the people you’ve trained,” Yuspa said. “Part of my

job as a scientist, mentor, and member of

the NIH is to help anybody I can to succeed.” 

About 36 postdocs have moved

through his lab. “I couldn’t be more proud of

the people who’ve trained here. They’ve

been uniformly successful and most [30]

have stayed in the same field,” Yuspa

beamed. 

“The reason that many of us have 

continued to work in science is that his

enthusiasm for science is infectious,” said

Dennis Roop, Ph.D. “When we trained with

him, we could see how much he enjoyed 

science. He hasn’t lost that. He’s still like a little kid when he starts talking about science.” 

Adam Glick, Ph.D., called Yuspa the

“grandfather of the field.” Go to any of the

A Generous Spirit 

2 8 ccr connections | v OLUME 2, N O . 1 | 2008

learning new things about cancer genes that

tell us about skin cancer and other kinds of

epithelial cancers, such as prostate, liver,

and kidney cancers.” 

“Many of the events that Stu has identified in the mouse have been confirmed as

also being causal genetic alterations that

result in human tumors,” said Dennis R.

Roop, Ph.D., who worked with Yuspa at NCI

in the 1980s and is now Chair of Regenerative

Medicine and Stem Cell Biology at the

University of Colorado Health Sciences

Center. “His early studies in the mouse provided the groundwork for understanding

that after a stem cell is initiated, it can sit for

a long time, then get an accumulation of

other genetic events to lead that initial 

epidermal stem cell to result in a tumor.”

The epidermis, where the keratinocytes live, is a hotbed of cell turnover,

Roop explained. This outer layer of our skin

replaces itself every three to four weeks in

humans. “Yet many of us develop skin cancer

late in life. Only the stem cells that reside for

a lifetime are around to accumulate enough

genetic damage to lead to cancer.” 

Roop added, “Stu was also really the

first to identify mutations in the Harvey ras

gene [a proto-oncogene known for its connections to skin and bladder cancers] as a

complete initiating event by showing that

just a single Harvey ras mutant gene in a keratinocyte [that is grafted onto an animal

model] would result in formation of a tumor.” 

“All of us have learned so much from

the work Stu started and others who’ve 

further developed the model,” KuleszMartin added. 

A Physician’s Dream 

“It’s everyone’s dream,” said Yuspa, “particularly if you’re a physician who’s done basic

science for the last 30 years, to translate

your discoveries into treatments for

patients.” To do that, Yuspa’s team is focusing on two relatively new discoveries. Using

a mouse model of induced skin inflammation, they are studying how inflammation

influences the frequency with which skin

cancers develop. In this model, skin cancer

is much more frequent. The group identified

a receptor that seems to be essential for

tumors to develop. He hopes that it could be

a novel target for preventing or treating skin

cancer.

The second discovery builds on an

insight made nearly 10 years ago. They have

shown that inactivation of the tumor suppressor gene p53 is important for malignant

feature

big meetings, he noted, and everyone is

somehow connected to Yuspa, either as collaborators, postdocs, or postdocs of his

postdocs. 

Replying wryly that he’d rather be

called the “father of skin carcinogenesis,”

Yuspa agreed that his proudest moments in

science occur at meetings when he sees his

protégés and theirs presenting important and

interesting information. “It feels very good.”

“Once you go to Stu’s lab, you become

part of a huge network of people from

around the world who are doing top-rate 

science,” said Molly Kulesz-Martin, Ph.D.

“We’re all excited about the skin and the science of the skin.”

And these “Yuspa graduates” open

their arms to the new people who continue

to come through the lab. “We share with anybody who asks. It’s a trait of Stu’s that we

learned at his knee,” Kulesz-Martin continued. “I remember talking to Stu about how

competitive science is and how people don’t

want to share. He said, ‘I’ve always shared

data. Maybe sometimes you get burned, but

I always learn something new when I share.’” 

Molly Kulesz-Martin, Ph.D.

Adam Glick, Ph.D.

(Photo: Courtesy of M. Kulesz-Martin) (Photo: Courtesy of A. Glick)
ccr connections | v OLUME 2, N O . 1 | 2008 2 9

feature

“It’s everyone’s

dream,” said

Yuspa, “particularly

if you’re a 

physician who’s

done basic science

for the last 30

years, to translate

your discoveries

into treatments for

patients.” 

conversion, a late event in carcinogenesis in

the skin. Using keratinocytes from p53-null

mice, they discovered one of p53 ’s transcriptional targets, a gene called chloride intracellular channel 4 (CLIC4). CLIC4's gene

product, CLIC4, was originally described as a

chloride channel protein but, as it turns out,

is multifunctional, being involved in cellular

responses to stressors such as DNA damage,

metabolic inhibition, and senescence.

Yuspa’s team found that, in mouse skin cancer and most major human cancers, CLIC4 is

lost from tumor cells. The protein is essential for cell death induced by p53. They are

trying to reactivate the expression of CLIC4

in tumor cells—not an easy feat. “We know

the gene is there; it’s not lost, just silenced

in some way,” said Yuspa. They are hopeful

they can unsilence the gene, reactivate

CLIC4 expression, and have some influence

on tumor cell growth.

Other pathways are standing out as

significant players as well. Yuspa’s group has

identified a pathway through protein kinase

C delta (PKCdelta)—a regulator of many 

different signaling pathways studied in

numerous solid and hematologic cancers—

that is important to the development of skin

cancer in mice. In an animal model, Yuspa

has been able to stimulate PKCdelta in tumor

cells and cause the tumor cells to regress,

activating a cell death (or apoptosis) pathway. 

An Australian company named Peplin

is developing a natural product drug that

works through a similar mechanism. In clinical trials, this drug appears to do the same

thing to PKCdelta in humans that the Yuspa

lab has done in animals. NCI has a cooperative research agreement with the company

to sort out the pathways involved in the

death of the tumor cells. 

Looking More than Skin Deep

Yuspa’s group continues to make exciting 

discoveries today. A recent study with Adam

Glick, now an Associate Professor in the

Center for Molecular Toxicology and

Carcinogenesis at Pennsylvania State

University, has ramifications for early detection and prevention of skin and other cancers. 

In May 2007, Yuspa, Glick, and colleagues from the American University of

Beirut reported that they had found an 

87-gene expression pattern, or genetic 

signature, in mice that distinguishes benign

tumors with a high or low risk of becoming

malignant. In particular, the high-risk lesions

had reduced expression of immune function

genes compared with low-risk lesions, a

finding Yuspa calls a “surprising, important

component of the study. High-risk tumors

may in some way be protected from surveillance by the immune system.” 

Now they will have to determine how

those findings can apply to human cancer—

and moving from mouse to human is neither

easy nor automatic. Human skin cancers are

induced by ultraviolet light, while the

tumors in Yuspa’s animal models are

induced by chemical carcinogens or oncogenes. As a result, the genetics may differ.

“But,” Yuspa added, “we feel that the pathways

that are downstream from the inducing

agents are very similar.” 

Knowing that skin cancer takes different forms, Yuspa has also started using his

models to investigate different cells. He has

initiated a collaboration with Glenn Merlino,

Ph.D., his Co-Chief at the LCBG, who studies

melanoma. Since both melanomas and

squamous cell cancers occur in the skin, and

there is an intimate relationship between

keratinocytes and melanocytes in the progression of melanoma, “bringing the two

models together will give us great insight

into melanoma’s induction,” said Yuspa. 

He has also pulled in partners to

make a difference in internal organ cancers

as well. “I’ve convinced [LCBG Adjunct

Investigator] Jonathan Wiest, Ph.D., to look

at skin as a model for lung cancer,” Yuspa

said. With Adam Glick, he’s hoping to look at

head and neck cancers. 

“These are all areas where we think we

can go beyond boundaries,” he said. 

( P h oto: Co u rtes y of D. Roo p )

Dennis Roop, Ph.D. (left), presents Stuart

Yuspa, M.D. (right), with the Stephen

Rothman Memorial Award for Investigative

Dermatology in 2004. The Rothman Award is

the highest award given by the Society to

members who have made outstanding contributions to investigative cutaneous biology.
The true power of a team can only

come from listening and interacting

with other researchers, clinicians, and

advocates, all committed to advancing

research and helping patients.

3 0 ccr connections | v OLUME 2, N O . 1 | 2008

As a high school student growing up in

Potomac, Md., near the National Institutes

of Health, I attended a program at the

National Naval Medical Center. While

there, I was drawn to biology, an interest

that shifted to molecular biology during my

undergraduate and medical studies.

Through a series of serendipitous events, I

spent part of my undergraduate years working part time in a laboratory that was

focused on liver cancer, an experience that

sparked my interest in cancer.

As a student at Harvard Medical

School, I again found myself working in a

lab during the summer, this time in a breast

cancer laboratory at NCI. It was here that

serendipity yielded to passion and planning. That summer was life-defining for me,

as it helped me realize that I wanted to

focus on breast cancer. This realization not

only gave me a clear path for my training,

but it ultimately led me back to NCI as a

Medical Staff Fellow from 1982–1985, and

then to Johns Hopkins, where I have been a

faculty member for over 20 years.

Good science is generally a combination

of reason and providence, and translational

breast cancer research requires a mix of

laboratory insight and clinical observation.

Investigation at the interface between the

bench and bedside drives my research,

even today, and it was a key factor in my

development as an independent physicianscientist. Early in my career, I was given the

opportunity to lead a large NCI-sponsored

clinical trial focused on premenopausal

women with breast cancer. At this critical

early time, I was—and still am—fortunate

COMMENTARY

to participate in the cooperative group

process as a committee member and chair

involved in the conduct of a number of clinical trials that examined optimal treatment

for women with breast cancer. These collaborations have helped advance the standard

of care in breast cancer by establishing

appropriate kinds of chemotherapy and

hormone treatments, and they instilled in

me a lasting fascination with the concept of

hormones in breast cancer and why some

types of cancer do not respond to estrogenrelated therapies while others do.

In the world of scientific research, methodical approaches, driven by logic, reign. Some of the most influential and 

lasting contributions to science, however, are the result of unexpected twists in experiments, serendipitous “accidents.”

Penicillin, for example, was discovered in 1928 by Alexander Fleming after he neglected to properly clean bacteria 

cultures and left them in his lab. The renowned 19th century chemist and biologist Louis Pasteur once said, “Chance

favors the prepared mind.” Rather than relying solely on methods or on chance, however, truly visionary scientific 

discovery can only be achieved through a balance of both. Nancy E. Davidson, M.D., former Medical Staff Fellow at

CCR, now President of the American Society of Clinical Oncology and Director of the Breast Cancer Research

Program at Johns Hopkins University’s Sidney Kimmel Comprehensive Cancer Center, offers her thoughts on how

that balance has affected her career as a breast cancer clinician and researcher.

A Series of Fortunate Events
ccr connections | v OLUME 2, N O . 1 | 2008 3 1

In addition to my early clinical research

experiences, the collaborative environment

of the laboratory also influenced my work.

When I first joined the faculty at Johns

Hopkins, my research centered on the roles

of oncogenes in breast cancer growth. A laboratory colleague of mine at the time, Johns

Hopkins scientist John Isaacs, Ph.D., pointed

out a disconnect between my research focus

and my clinical practice. “As a physician,” he

noted, “you work with patients to destroy the

cancer invading their bodies. But as a

researcher, you devote your time to discovering how to make breast cancer cells grow.” 

This chance conversation had a transformative effect on my research. I turned

from studying the proliferation of breast

cancer cells to studying their death, 

refocusing my work on apoptosis in breast

cancer cells. Over time, my focus shifted

again to bring my laboratory research back

in line with my abiding clinical interest in

the unique biological connections between

hormones and breast cancer. 

These connections have been a common theme in the field for many years. The

estrogen receptor (ER) has long been the

target of therapies aimed at reducing the

growth-promoting influence of estrogen on

breast cancer cells; the first hormone therapies for breast cancer were actually used

over 100 years ago. While this is not a new

arena for us, it is kept vital by the strides

that researchers have made in recent years

toward understanding the biology of hormones and breast cancer and its clinical

exploitation. 

But about 25 percent of human breast

cancers lack ER expression, a trait that

makes them resistant to hormonal therapies. Our current research focus on the 

epigenetics of ER expression is aimed at

understanding one mechanism by which

ER expression may be silenced in some

breast cancers. With this knowledge in hand,

we can work to develop a way to reverse this

process, perhaps making it possible to treat

these endocrine-resistant forms of cancer

with traditional hormonal treatments. 

As a new breast cancer researcher in

the 1980s and 1990s, I had the good fortune

to enter the field in a time of great change

and promise, a feeling that persists even

today. The advent of modern molecular

biology techniques, advances in our knowledge of the human genome, engagement of

breast cancer patients, and a shift toward

very scientifically-based clinical trials

should put us even closer to achieving our

goal of reducing the burden of cancer.

This optimism, however, is tempered

by the reality of the research and healthcare

environment that both researchers and 

clinicians face on a daily basis. Though we

have made a number of inroads, we are still

confronted by challenges that make it difficult to take advantage of these opportunities

in the current research and clinical climates.

Reduced research funding, increasing

healthcare costs, and a population of

approximately 47 million uninsured

Americans1 make it more difficult to use

our growing knowledge about cancer to

improve the well-being of all patients. 

To overcome these research and clinical challenges, it is important that we not

only remain open to those serendipitous

moments that present themselves but also

utilize them in the most efficient way possible. In addition, we must recognize the 

benefit of collaboration in our work. The

true power of a team can only come from

listening and interacting with other

researchers, clinicians, and advocates, all

committed to advancing research and helping patients.

As a researcher, I am ever more interested in working at the interface between

the lab and the clinic. My lab science grows

more reflective of questions that I see in

the clinic, and I like to think that my clinical

research and clinical practice are ever more

driven by good science and rigorous evidence. Of course all of the techniques and

methods used in breast cancer research can

be applied to different types of cancer as

well. I believe that all cancer researchers

can make focused and practical choices

while staying receptive to the unexpected

moments that can help drive truly beneficial science forward. 

1

National Coalition on Health Care. “Health Insurance

Coverage.” http://www.nchc.org/facts/coverage.shtml. Accessed

February 27, 2008.

Nancy E. Davidson, M.D. 

Breast Cancer Research 

Professor of Oncology 

Johns Hopkins University 

School of Medicine

Baltimore, Md.

COMMENTARY

(Photo: Courtesy of N. Davidson)
3 2 ccr connections | v OLUME 2, N O . 1 | 2008

The discovery by 

Luc Montagnier and

Robert C. Gallo of 

a novel human 

retrovirus, and Gallo’s

demonstration that

this virus was the

cause of AIDS, were

the first major 

milestones in our

efforts to understand,

prevent, and treat 

the disease. 

surfaced. Though heroic measures were

often utilized to try to treat these patients,

they generally died within several months. 

It was not known how many people had this

disease, how it was transmitted, or if anything could be done to treat it effectively.

The NCI had many tools in place to

address this new disease, including expertise

in immunology, retrovirology, tumor biology,

and drug development. I believe that the

rapid development of advanced treatments

for HIV, in particular highly active antiretroviral therapy (HAART), was the result of a

keen and farsighted, though sometimes controversial, research focus at NCI on HIV itself.

To Treat a Virus 

The discovery by Luc Montagnier and Robert

C. Gallo of a novel human retrovirus, and

Gallo’s demonstration that this virus was the

cause of AIDS, were the first major milestones in our efforts to understand, prevent,

and treat the disease. Prior to these discoveries, most of the research on AIDS was

descriptive. 

Once the scientific community understood what was causing the immunodeficiency, it was possible to envision effective

therapy directed at the root cause. Then-NCI

Director Vincent DeVita, Jr., M.D., asked

Samuel Broder, M.D., and his group, of which

I was a member, to spearhead an effort 

to develop treatments for AIDS. Broder, 

Hiroaki Mitsuya, M.D., Ph.D., now Head of

the Experimental Retrovirology Section of

the HIV and AIDS Malignancy Branch, and I 

constructed the hypothesis that by blocking

HIV replication, it might be possible to

reverse the immunodeficiency caused by the

virus. This idea was somewhat controversial

at the time, as no such therapies existed for

other progressive viral diseases, and it was

An estimated 35 million people suffer from

HIV/AIDS worldwide, about one million of

whom live in the United States. However,

when the first patient with AIDS crossed the

threshold of the NIH Clinical Center in 1981,

we had no way of foreseeing the shape that

the future AIDS epidemic would take or the

global, cultural, and societal impacts that it

would have. All we knew was that we were

seeing something new. It was soon evident

that clusters of cases of rare tumors like

Kaposi’s sarcoma were part of this same

condition. And over several months, it

became apparent that that this disease was

widespread in the U.S., and that the more

severe patients with AIDS we were seeing

represented just the tip of the iceberg.

There was great confusion and distress in the medical community as AIDS first

Combating HIV, 

One Tumor at a Time

Robert Yarchoan, M.D., vividly remembers the day in 1981 when, as a

Clinical Fellow in the Metabolism Branch of NCI, he saw a young man

who had developed a profound immunodeficiency. That patient, who had

almost no T lymphocytes, represented a moment in medical history—

the first NIH patient with what would come to be known as acquired

immunodeficiency syndrome (AIDS). Since that time, Yarchoan, now

Chief of the HIV and AIDS Malignancy Branch and Director of the

newly formed NCI Office of HIV and AIDS Malignancy (OHAM), has

seen firsthand how HIV/AIDS has risen as a global epidemic, how the 

development of highly active antiretroviral therapy (HAART) has revolutionized AIDS treatment, how malignancies associated with AIDS have

tested both patients and doctors, and how the long-term effects of living

with AIDS can bring with them a new host of challenges. 

IN THE CLINIC
ccr connections | v OLUME 2, N O . 1 | 2008 3 3

Mitsuya and I were honored in 2006 with the

first NIH World AIDS Day Award (Figure 2).

The first-generation protease inhibitors,

while effective, had significant side effects

and required high doses to be effective.

Moreover, the development of resistance has

been a vexing problem. Therefore, a number

of scientists (including Mitsuya) and pharmaceutical companies have been looking

into the development of improved, secondgeneration protease inhibitors. Darunavir,

developed by Mitsuya and collaborators and

approved by the U.S. Food and Drug

Administration in 2006, is one such drug.

Darunavir has the advantage of retaining its

activity even in patients who have become

resistant to other inhibitors. 

For several years, my colleague David

Davis, Ph.D., and I have been studying the

possibility of inhibiting HIV protease by a

new mechanism: by blocking the linkage of

its two monomers into an active dimer.

Working with the two of us, Mitsuya recently

found that in addition to blocking the

enzyme’s active site, darunavir does just this,

making the drug quite different from other

approved HIV protease inhibitors. This

observation was highlighted as one of CCR’s

major scientific advances of 2007 (see

“Multiple Strategies for Attacking HIV”).

The Tumors of AIDS

One of the hallmarks of HIV is a dramatic

increase in the incidence of certain unusual

cancers, especially Kaposi’s sarcoma and

certain aggressive B-cell lymphomas.

Kaposi’s sarcoma was frequently the cause

of death for patients in the early days of 

the AIDS epidemic. This rare sarcoma—

which, prior to the rise of HIV/AIDS, had 

primarily been known as a cancer of elderly

men of Mediterranean or Eastern European

Jewish descent—develops in the endothelial

cell lining of blood vessels and often affects

not clear that blocking HIV would lead to

immunologic improvement. In a short time,

our group in NCI developed the first therapies

to effectively fight HIV infection, including

zidovudine (AZT), didanosine (ddI), and zalcitabine (ddC). AZT was developed in collaboration with Burroughs Wellcome Co., while

ddI and ddC were developed within NCI and

then licensed to pharmaceutical companies.

These drugs all blocked an essential and

unique enzyme of HIV, reverse transcriptase.

The initial clinical trials of these drugs, both

singly and in combination, gave rise to the

first effective anti-HIV treatment regimens. 

A limitation of these initial regimens

was the frequent development of viral resistance. Thus, about a decade later, the first

drugs were developed that attacked a different HIV target, the viral protease. The creation

of these so-called protease inhibitors, which

block the active site of HIV protease, was in

part based on structural studies of this

enzyme conducted in the NCI-Frederick program. When combined with two nucleoside

reverse transcriptase inhibitors (like AZT and

ddI), protease inhibitors were able to suppress HIV replication dramatically, often to

undetectable levels. This combination drug

therapy, the backbone of HAART, revolutionized the treatment of HIV. Patients improved

clinically and had marked improvement in

their immune function. There was a dramatic

drop in the death rate from AIDS. In addition,

there was a marked decrease in the number

of AIDS-associated tumors, and survival of

patients with these tumors improved. For our

contributions to the development of HAART,

(Photo: Digital Micrograph, Gatan Inc.)

Figure 1: Tireless work on the part of researchers at NCI and other institutions 

has led to dramatic decreases in the mortality associated with HIV/AIDS and

AIDS-related malignancies. But challenges still remain.

This combination

drug therapy, 

the backbone 

of HAART, 

revolutionized

the treatment 

of HIV patients.
3 4 ccr connections | v OLUME 2, N O . 1 | 2008

the skin, though it may also involve the

mouth, gastrointestinal tract, and lungs. The

tumors appear as angry red and purple

patches, often on the legs and feet. These

lesions are a visible manifestation of HIV disease, often causing substantial psychological

distress for patients in addition to severe

pain and debilitation. 

The cause of Kaposi’s sarcoma

remained a mystery until 1994, when Yuan

Chang, M.D., and Patrick Moore, M.D., M.P.H.

(now at the University of Pittsburgh), discovered a new gammaherpesvirus and showed it

to be the cause of Kaposi’s sarcoma. We now

know that this virus, called Kaposi’s sarcomaassociated herpesvirus (KSHV) or human

herpesvirus-8 (HHV-8), is also the etiologic

agent of two other tumors seen in AIDS

patients—primary effusion lymphoma (PEL)

and some cases of multicentric Castleman’s

disease (MCD).

After focusing initially on treatments

for HIV, my group started turning its 

collective attention to AIDS-associated

malignancies. In the early 1990s, we showed

that paclitaxel (Taxol®) was active against

Kaposi’s sarcoma. Then, since Kaposi’s 

sarcoma is a cancer of blood vessels, we

began exploring anti-angiogenic approaches

that would interfere with blood vessel

growth. We subsequently showed that

thalidomide, a strongly anti-angiogenic

agent, was also active against the tumor. 

More recently, my colleagues James

Pluda, M.D., Giovanna Tosato, M.D., and

Richard Little, M.D., and I have turned our

attention to the cytokine interleukin-12 (IL12), a powerful immune system regulator, as

a possible treatment for Kaposi’s sarcoma.

We believed that there were at least three

ways in which IL-12 could act against

Kaposi’s sarcoma:

• It could enhance the immune response

against KSHV.

• It could act as an anti-angiogenic agent,

shrinking the cancer’s heavily vascularized

tumors.

• It could work through another protein, 

a chemokine called inducible protein-10

(IP-10), which inhibits a virally encoded 

G-protein-coupled receptor that is important

for the pathogenesis of Kaposi’s sarcoma. 

We carried out a clinical study, published in

2006, showing that IL-12 was active against

Kaposi’s sarcoma when used as a single

agent. Subsequently, we decided to see if

more rapid and complete responses could

be attained by combining IL-12 with a cytotoxic agent, pegylated liposomal doxorubicin

(or Doxil®), with known activity against

Kaposi’s sarcoma. 

This research led to the launch of a

Phase II clinical trial, spearheaded by Little,

in 36 patients with advanced AIDS-associated

Kaposi’s sarcoma who were either on HAART

or needed urgent therapy. All of the patients

received IL-12 plus Doxil for 18 weeks, followed by IL-12 alone.

Thirty of the patients demonstrated

significant responses, including nine 

complete remissions. Most of the patients

maintained, or even improved, their condition while on the IL-12 only portion of the

study, so we published the results in

December 2007. While this regimen has yet

to be tested in a controlled trial, the uncontrolled results are quite promising. 

Coming to the Foot of the Matter

Working within CCR provides physicianscientists exceptional opportunities to 

conduct translational research—studies that

can be viewed as a two-way street between

the laboratory and the clinic. For some time,

clinicians treating Kaposi’s sarcoma had

been struck by the tendency of this tumor to

preferentially develop in certain parts of the

body, particularly the feet. The feet are 

relatively poorly oxygenated, raising the 

possibility that hypoxia might have some

connection to the growth and development

of Kaposi’s tumors. Later, in discussing this

finding with colleagues, it dawned on us that

PEL, another of the tumors caused by KSHV,

also formed in a site with limited oxygenation (pleural effusions, accumulations of

fluid within the chest cavity).

In thinking about these peculiarities with

my colleagues, we considered the possibility

that KSHV might somehow be responsive to

hypoxia. Like other herpesviruses, KSHV can

undergo either latent or lytic replication. My

group, including Davis and Muzammel

Haque, Ph.D., found that in PEL cell lines the

virus was activated to lytic replication by

hypoxia, and that certain specific genes of the

virus could directly respond to hypoxiainducible factors (HIF) produced by host

cells. This was the first time that any virus

had been shown to respond to hypoxia or

HIF, and it gave great insight into the pathogenesis of KSHV-induced tumors (see “Why

Do Kaposi’s Sarcoma Lesions Most Often

Develop on the Feet?”).

This observation led us to consider a

novel therapeutic approach for KSHVinduced associated tumors. When activated

by hypoxia or other factors, KSHV expresses

two lytic genes that encode enzymes capable

of catalyzing AZT and another antiviral drug,

ganciclovir, into forms that are toxic to

human cells. We reasoned that this capability

could be used to target tumors caused by

KSHV, in particular PEL and MCD, a disease

marked by serious, sepsis-like illness caused

in part by the production of a form of the

cytokine interleukin-6 encoded by KSHV

IN THE CLINIC

(Gr a p h : R. Ya rc h o a n , CCR)

Figure 2: The development of highly active antiretroviral therapy (or HAART) has

been the driving force behind a revolution in the treatment of HIV/AIDS.
ccr connections | v OLUME 2, N O . 1 | 2008 3 5

IN THE CLINIC

itself. MCD is also unique among KSHVrelated diseases because the virus’s lytic

genes are already activated. 

Together with Little and Deirdre

O’Mahony, M.D., we are investigating the use

of these two drugs in patients with MCD in a

unique clinical trial that is also helping us to

further understand the disease’s natural history. This trial plays to one of CCR’s main

strengths: its ability to recruit patients from

around the nation and study very rare diseases. Thus far, about half of the MCD

patients treated with high doses of AZT and

valganciclovir (a prodrug of ganciclovir) have

responded. The trial involves a collaboration

of many groups within NCI and the NIH that

we hope will lead to a better understanding

of MCD’s pathology and responses to therapy; in the future, we hope to explore this

approach in patients with PEL as well. But

this chain of events—from the clinical observation of Kaposi’s anatomical distribution, 

to the discovery of KSHV’s hypoxic 

elements, to a novel therapeutic option for

MCD—is a prime example of the how clinical

observation can lead to laboratory insights

that then lead back to the clinic to inform the

design of a new treatment. 

Long-Term Survival, 

Long-Term Challenges, 

and Long-Term Opportunities

The development of HAART has arguably

been the greatest advance in the prevention

and therapy of AIDS-related tumors. One of

the major victories of HAART has been a

reduction in the incidence of certain AIDSassociated malignancies, especially those

that develop in patients with markedly

reduced CD4 counts. Patients are living for

years on HAART, compared to the weeks or

months that they might have hoped for 20

years ago. 

However, as we enter the second

decade of HAART therapy, the epidemiology

of AIDS-related tumors and diseases is

changing (Figure 3). In some patients,

Kaposi’s sarcoma tumors that had been

under control for years are beginning to grow

again. Also, tumors other than those “classically” defined as AIDS-related, such as

Hodgkin’s disease, lung cancer, and anal carcinoma, are on the rise in the HIV-infected

population. The number of deaths from

opportunistic infections and advanced AIDS

is dropping, while cancer is fast becoming

the most common cause of death in AIDS

patients. In the meantime, in Africa and

other parts of the world where HAART is not

commonly available, HIV-associated tumors

continue to be major causes of morbidity

and mortality.

These new trends, as well as the new

As we enter the 

second decade of

HAART therapy, the

epidemiology of

AIDS-related tumors

and diseases is

changing. 

(Gr a p h : R. Ya rc h o a n , CCR)

Figure 3: The epidemiology of AIDS-related tumors and illness is changing as 

the number of AIDS patients in the United States grows. This represents an

increasing population at risk of developing malignancies.

Robert Yarchoan, M.D.

opportunities afforded by recent scientific

advances, will require a renewed research

effort by the National Cancer Institute. To

this end, the NCI Director has recently created a new Office of HIV and AIDS

Malignancy (OHAM) that will coordinate

AIDS and AIDS malignancy research

throughout the institute. At the same time,

CCR has launched a new Center of

Excellence in HIV/AIDS and Cancer

Virology, led by Stuart Le Grice, Ph.D. With

the renewed interest in this area and the

creation of these two groups, the NCI is

poised to have a substantial impact on

AIDS malignancy and AIDS research on a

global scale.

(Photo: B. Branson)
While highly active antiretroviral therapy

(HAART) is able to keep levels of HIV very low

and deter the onset of AIDS, it does not 

provide a cure. Lingering viral reservoirs 

persist undectable in the blood and other

tissues, ultimately enabling HIV to develop

resistance to antiretroviral drugs and begin

propagating despite HAART. The identification

of new types of drugs that attack HIV in 

different ways might help keep these drugresistant viruses in check. 

A group led by Hiroaki Mitsuya, M.D., Ph.D.,

recently developed a new antiretroviral called

darunavir, which was specifically designed to

tightly bind and inhibit the HIV protease.

Darunavir is more potent than most available

first-generation protease inhibitors (PIs), and it

can block replication of HIV strains resistant to

multiple other PIs. 

Mitsuya recently teamed up with his longstanding collaborator Robert Yarchoan, M.D., and

other researchers to learn more about how

darunavir inhibits HIV protease. The HIV protease

is made up of two protein monomer subunits that

must come together, or dimerize, to create the

mature, active form of the enzyme. It is the mature

protease that plays a critical role in HIV replication. 

Mitsuya, Yarchoan, and colleagues found

that darunavir can work just like other PIs that have

been used for years—by preventing the mature

protease from processing other HIV proteins.

However, they discovered that darunavir has

another trick up its sleeve—it is also able to prevent the two parts of the protease from dimerizing.

This study marked the first time that a small molecule was found to disrupt protease dimerization.

Because it inhibits HIV protease using a unique

mechanism (i.e., inhibition of protein dimerization),

darunavir—and possibly other molecules like it—

may be useful for treating individuals infected with

HIV that have developed resistance to more 

traditional PIs and other antiretroviral therapies. 

In his original description of Kaposi’s sarcoma in 1872, Moritz Kaposi noted that the

purple, brown, or black lesions of the condition tended to occur on his patients’ feet.

Over time, researchers and physicians alike

have been struck by a predilection of

Kaposi’s to involve the feet and other areas

of the body with a poor blood supply. It has

been speculated that this tendency is

because the legs and feet often have relatively low tissue-oxygen levels (hypoxia).

Interestingly, another tumor caused by

Kaposi’s sarcoma-associated herpesvirus

(KSHV), primary effusion lymphoma (PEL),

arises in pleural effusions—accumulations

of excess fluid in the chest with no direct

blood supply and, consequently, poor oxygenation.

Generally, after initial infection, herpesviruses enter a dormant or resting phase,

allowing them to remain within the host for

years undetected only to arise when stimulated by various factors. This stimulation to

lytic replication—which results in the rupture of the host cell—generally involves one

or more switch genes, which in turn activate

a cascade of viral genes.

In exploring KSHV-induced tumors’

tendency to arise in hypoxic areas, Robert

Yarchoan, M.D., and his colleagues found

that lytic replication KSHV in PEL cell lines

is activated in hypoxic cells. In addition, the

team found that certain specific KSHV genes

could be directly activated by hypoxia,

including a cluster of lytic genes stretching

from ORF34 to ORF37. Cells exposed to

hypoxia express increased levels of so-called

hypoxia-induced factors (HIFs). Yarchoan’s

group found that the ORF34–37 gene cluster

could be directly stimulated by the binding

of HIFs to a single viral hypoxia response

element (HRE) located in the cluster’s promoter region. Because some of these KSHV

genes play important roles in the pathogenesis of KS or other tumors, their hypoxic

activation could thus help explain why these

tumors arise where they do.

His group is now exploring the possibility that the activation of certain KSHV genes

by hypoxia could be used as a means to treat

tumors caused by KSHV. Two hypoxiainduced viral genes, ORF21 and ORF36, can

chemically modify the drugs ganciclovir and

zidovudine (AZT) into forms toxic to cells.

Yarchoan is now testing whether these two

drugs can be used to specifically target KSHVinduced tumors in which ORF21 and ORF36

are activated by hypoxia or other means.

Multiple Strategies

for Attacking HIV Why Do Kaposi’s Sarcoma

Lesions Most Often Develop

on the Feet?

IN THE CLINIC

3 6 ccr connections | v OLUME 2, N O . 1 | 2008

Because of hypoxia-responsive genes encoded by the virus that causes Kaposi’s 

sarcoma, the lesions of this tumor have a tendency to show up in relatively poorly

oxygenated tissues, like the feet.

Hiroaki Mitsuya, M.D., Ph.D. 

(Photo: NCI)

(Photo: B. Branson)