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Leukemia (2003) 17, 2358–2382
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REVIEW
Treatment by design in leukemia, a meeting report, Philadelphia, Pennsylvania,
December 2002
RA Larson1, GQ Daley2, CA Schiffer3, P Porcu4, C-H Pui5, J-P Marie6, LS Steelman7, FE Bertrand7 and JA McCubrey7,8
1
Section of Hematology/Oncology, University of Chicago Pritzker School of Medicine, Chicago, IL, USA; 2Whitehead Institute for
Biomedical Research and Harvard Medical School, Cambridge, MA, USA; 3Wayne State University School of Medicine,
Karmanos Cancer Institute, Detroit, MI, USA; 4Division of Hematology Oncology, Ohio State University College of Medicine
and Public Health, Columbus, OH, USA; 5St Jude Children’s Research Hospital and the University of Tennessee Health Science
Center, Memphis, TN, USA; 6Onco-Hematology Department, Hôtel-Dieu de Paris, Paris, AP-HP, France; 7Department of
Microbiology and Immunology, The Brody School of Medicine at East Carolina University, Greenville, NC, USA; and 8Leo Jenkins
Cancer Center, The Brody School of Medicine at East Carolina University, Greenville, NC, USA
Novel approaches have been designed to treat leukemia based
on our understanding of the genetic and biochemical lesions
present in different malignancies. This meeting report summarizes some of the recent advances in leukemia treatment.
Based on the discoveries of cellular oncogenes, chromosomal
translocations, monoclonal antibodies, multidrug resistance
pumps, signal transduction pathways, genomics/proteonomic
approaches to clinical diagnosis and mutations in biochemical
pathways, clinicians and basic scientists have been able to
identify the particular genetic mutations and signal transduction pathways involved as well as design more appropriate
treatments for the leukemia patient. This meeting report
discusses these exciting new therapies and the results
obtained from ongoing clinical trials. Furthermore, rational
approaches to treat complications of tumor lysis syndrome by
administration of the recombinant urate oxidase protein, also
known as rasburicase, which corrects the biochemical defect
present in humans, were discussed. Clearly, over the past 25
years, molecular biology and biotechnology has provided the
hematologist/oncologist novel bullets in their arsenal that will
allow treatment by design in leukemia.
Leukemia (2003) 17, 2358–2382. doi:10.1038/sj.leu.2403156
Published online 9 October 2003
Keywords: leukemia therapy; signal transduction inhibitors;
chromosomal translocations; multidrug resistance; monoclonal
antibodies; rasburicase; elitekt; tumor lysis syndrome
Introduction and overview of meeting: Richard A Larson, MD
Several decades of prospective clinical trials have allowed the
identification of important determinants of response for patients
with leukemia that correlate with both treatment and survival.
These have included age, the presence of comorbid illnesses,
and the number of white blood cells (WBC) at time of diagnosis,
and precise morphological diagnoses. Recently, more sophisticated techniques have been used to examine the biological
characteristics of the leukemia itself including immunophenotyping, karyotyping and gene expression. This knowledge has
led to risk-adapted treatment plans in which the use of particular
cytotoxic drugs and supportive care measures have been
designed for individual patients. Empiricism is gradually being
replaced by individualized therapy.
Targeted therapy did not start with STI571, now known as
imatinib mesylate or Gleevect. The possibility of curing
Correspondence: JA McCubrey, Department of Microbiology and
Immunology, The Brody School of Medicine at East Carolina
University, Brody Building 5N98C, Greenville, NC 27858, USA;
Fax: þ 1 252 744 3104
Received 1 August 2003; accepted 26 August 2003; Published online
9 October 2003
disseminated cancer was ushered in approximately 50 years
ago, when investigators recognized that inhibiting folate
metabolism led to the death of childhood leukemia cells.
Methotrexate, which inhibits dihydrofolate reductase and other
enzymes, is still a cornerstone of treatment for acute lymphoblastic leukemia (ALL), although it has a minimal role in acute
myeloid leukemia (AML). However, methotrexate is not
selective. It inhibits the target enzymes in both normal and
malignant cells.
This symposium reviewed a number of pathways that are
known to be important in the regulation of cell proliferation,
apoptosis, drug resistance and metabolism in acute and chronic
leukemia. Promising new anticancer drugs and the current status
of clinical trials of investigational agents that have been
designed to specifically inhibit or alter these pathways and
interact with leukemia-specific targets were discussed at this
meeting.
This meeting report covers the role and requirement of the
BCR-ABL oncoprotein in chronic myelogenous leukemia (CML),
the transition from the chronic to the blast crisis stage and the
permissive role for additional chromosomal translocations
involving transcription factor genes and the roles of FLT-3 and
RAS mutations in leukemia. The treatment of CML and other
leukemias with Gleevect, and FLT-3 and RAS inhibitors is also
discussed. Certain chromosomal translocations involve the
expression of novel transcription factors that can exert dominant
negative (DN) effects on gene expression by silencing transcription through chromatin remodeling by tethering histone
decacetylases (HDAC). Treatment of certain leukemias with
retinoic acids and HDAC inhibitors may prove efficacious.
A frequent consequence observed in leukemia treatment with
common anticancer drugs is drug resistance. Hematopoietic
precursor cells may be intrinsically resistant to many drugs since
they were ‘designed’ to survive repeated exposure to heterogeneous toxins and natural products. Drug resistance can arise
by many different mechanisms; however, a common mechanism is increase in expression of a family of proteins called drug
transporters (P-glycoprotein (P-gp), MRP-1, breast cancer
resistance protein (BCRP) and others). These transmembrane
proteins contain an ATP-binding cassette (ABC) and are
associated with energy-dependent efflux of natural products.
Many antileukemia drugs are substrates for multidrug resistance
(MDR)-mediated efflux. P-gp, the protein product of the MDR-1
gene, is frequently overexpressed in stem cells. P-gp overexpression in AML is associated with a poorer prognosis. A
means to circumvent MDR is the use of MDR inhibitors.
Numerous MDR inhibitors have been developed and their
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RA Larson et al
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effects are being evaluated in clinical trials (cyclosporine, PSC833, LY335979, VX710, XR9575, R101933, PK11195 and
GG120918). The commonly used immunosuppressive drug
cyclosporine is known to reverse the effects of P-gp.
Chronic lymphocytic leukemia (CLL) is the most frequent
leukemia observed in the Western Hemisphere. Historically,
CLL has been treated with alkylating drugs. Now, however,
fludarabine is the best single agent for initial therapy. Chimeric
and humanized monoclonal antibodies (mAbs) represent novel
therapeutic options for CLL. The humanized mAb targeting
CD52 (CAMPATHs), a chimeric aCD20 Ab (Rituxans), an mAb
targeting CD23 (IDEC 152) and a humanized mAb targeting an
epitope of the HLA-DR b chain (Hu1D10, Remitogent) are in
various stages of clinical trials in CLL patients. These modified
antibodies may exert their cytotoxic effects by inducing
apoptosis.
ALL can be cured in approximately 80% of children and 30–
40% of adults. While up to 75% of ALL patients show
identifiable genetic abnormalities associated with prognostic
and therapeutic relevance, there remains considerable genetic
heterogeneity, which can complicate effective treatment. Some
high-risk ALL patients with certain chromosomal translocations
(eg, BCR-ABL and MLL-AF4) can be effectively treated with
allogeneic hematopoietic stem cell transplantation. The ability
to classify ALL by genomic/proteomic approaches represents a
significant advance in our understanding of the disease and will
eventually provide the clinician with more precise therapeutic
options for the patient.
Tumor lysis syndrome (TLS) is a serious and potentially lifethreatening complication of cancer chemotherapy, especially in
rapidly proliferating malignancies such as Burkitt’s lymphoma.
TLS involves an increase in the levels of uric acid, potassium,
phosphorous and other intracellular components due to the lysis
of tumor cells. This increase in uric acid and other metabolites
can lead to renal impairment primarily due to the insolubility of
uric acid in the urine. Humans lack the ability to metabolize uric
acid to allantoin, which is five times more soluble than uric
acid, due to a mutation in the urate oxidase gene. For the past 30
years, urate oxidase has been purified from Aspergillus flavus
and used to treat Burkitt’s lymphoma, ALL and AML patients in
France and Italy. Rasburicase is a recombinant urate oxidase.
Rasburicase is effective in preventing and treating hyperuricemia. Thus, this meeting covered various aspects of how basic
scientific discoveries over the past 30 years have been applied
clinically to improve significantly treatment of leukemia
patients.
Figure 1
Promising targets in leukemia: translating molecular
mechanisms into rational therapies: George Q Daley, MD
Dr Daley (Whitehead Institute and Harvard Medical School,
Cambridge, MA, USA) discussed some of the scientific basis for
rational drug design in leukemia. The dramatic success of
Gleevec has galvanized the drug development community
toward the identification of further such agents. This development has been facilitated by insights into the molecular
mechanisms of cancer.
There is an ongoing debate as to whether Gleevec will prove
to be a rare example of great success or whether the principles
responsible for Gleevec’s usefulness can be generalized to other
tumor types. It is likely that our understanding of the rather
limited set of pathways in cancer cells that lead to transformation can in many instances be targeted in the next decade or
two.
How many mutations are required to transform a normal cell
into a malignant cell? This fundamental issue has been studied
extensively in solid tumors by Vogelstein and colleagues at
Johns Hopkins University (Baltimore, MD, USA). By studying
the occurrence of various mutations in colon cancers, Vogelstein has derived a cancer model referred to by some as a
‘Vogelgram’, which assigns a sequence of stepwise mutations
across the spectrum of colon cancer. It is estimated that at least
four independent mutations must cooperate in the creation of a
full-blown colonic malignancy.1,2 A diagram representing this
concept is presented in Figure 1.
Weinberg and colleagues at MIT modeled that work in
primary cells,3 and recently it has become clear that at least
three or four genetic mutations are required for the malignant
transformation of primary cells. In initial rodent studies in the
1980s, it was shown that mutations at only two oncogenes myc
plus ras were needed to generate a tumor cell.4 However, when
those studies were attempted in human primary tissue, no
tumors arose. It was not until the isolation of telomerase and the
identification of its role in cellular immortalization did
malignant transformation experiments in human cells start to
work.5
Now it is clear that in primary human tissues, several
collaborating oncogenes are required for malignant transformation. These genes fall into general categories, biological pathways or functional pathways that need to be deranged in all
tumor cells in order for full tumorigenic phenotype to be
realized. Immortalization and prevention of apoptosis need to
be overcome. Genetic lesions that drive cell proliferation are
Multiple mutations and time are required for the transformation of a malignant colon cancer cell.
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RA Larson et al
2360
necessary, and ras pathway mutations are common. Also, the
problem of cellular senescence must be overcome and that is
the function of the telomerase gene product.
Standing in contrast to solid tumors, CML is an unusual
disease that is classified as a myeloproliferative disorder. It is a
weak malignancy, which some physicians consider a hyperplasia. The myeloproliferative phase of CML is associated with a
consistent chromosomal abnormality recognized as a balanced
translocation between distal elements of chromosomes 9 and
22.6 The human homologue of the Abelson murine leukemia
virus oncogene, c-abl, is located on chromosome 9 and gets
translocated and juxtaposed to a breakpoint cluster region on
chromosome 22. This results in the Philadelphia chromosome
(Ph), which encodes among other things the BCR-ABL oncoproteins.7
Over 10 years ago, Daley and colleagues at MIT inserted the
BCR-ABL oncogene into a mouse retrovirus.8 Bone marrow cells
of a mouse were infected with the recombinant retrovirus and a
bone marrow transplant was performed. A CML-like myeloproliferative condition in the recipient mouse was observed. This
led to the conclusion that BCR-ABL was indeed the causative
genetic lesion driving CML.
Leukemia
It took a decade, however, to make this process of retroviral
gene transfer and CML development more efficient, which
confirmed that BCR-ABL, acting as a single agent, was in fact
responsible for CML.9,10 It is known that every hematopoietic
stem cell that gets infected by the BCR-ABL virus will contribute
to the disease, and under experimental conditions in mice, a
polyclonal disorder is observed. This suggests that at least in
mice, the BCR/ABL oncoprotein alone can drive CML (Figure 2).
This concept has now been generalized. There are many
different chromosomal translocations that have been identified
and characterized in the context of these hyperplasia-like
myeloproliferative conditions. The translocation of 9 and 22,
which juxtaposes BCR-ABL, is the best characterized and the
most common, but it is now known that there are chromosomal
translocations such as fusion of PDGF receptor and other genes
such as TEL.11 TEL also fuses with ABL, and to other kinases like
JAK.11 In general, the phenomenon seems to be that an activated
kinase is responsible for driving the myeloproliferative phenotype. The global sites where these and other chromosomal
translocations may act in the cell are presented in Figure 3.
Genetic mutations that destroy the kinase activity of BCR-ABL
abrogate all transforming functions of this oncoprotein. This
Figure 2
Steps in transformation of human hematopoietic cells.
Figure 3
Conversion from chronic to acute myelogenous leukemia requires at least two mutations.
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RA Larson et al
2361
structure/function information provided the rationale for the
development of the small molecule inhibitor Gleevec, which is
a highly potent and specific inhibitor of the kinase activity
developed by Novartis. If CML patients are treated with Gleevec
immediately after diagnosis in the chronic phase, greater than
98% will achieve a complete normalization of the WBC
counts.12–14 The vast majority of patients achieve a major
cytogenetic response, that is, virtually complete suppression of
evidence of the Ph in their cells. Clearly this is a dramatic result.
The chronic phase of CML is unstable, and over time, these
patients, left untreated, will progress to a malignancy typical of
acute leukemia. This is called blast crisis. Blast crisis is the
consequence of additional genetic abnormalities. This has been
known at the cytogenetic level for many years, where patients
who evolve blast crisis have an extra Ph, abnormalities of
chromosome 17, trisomy 8, and a whole host of other
superimposed genetic events.
It thus appears that AML is at least a two-hit disease. This can
be modeled in experimental murine systems, and supporting
evidence has been gathered by cytogenetic analysis of clinical
specimens. Patients have been described who present initially
with the chronic phase of CML [t(9;22) bearing], and later
evolve into blast crisis with leukemia cells bearing secondary
genetic lesions such as the activation of homeobox genes or
core-binding factor (CBF). This has also been seen in patients
with myeloproliferative syndrome (MDS) characterized by the
(5;12) translocation that evolved into AML with an additional
(8;21) translocation.
A concept promulgated by Dr Gary Gilliland (Harvard
University, Boston, MA, USA) is that myeloproliferative conditions seem to evolve from activated kinases, whereas the
progression to acute leukemia requires a block in differentiation
that is typically seen in the context of translocations, which alter
transcription factors.15 This concept has also been modeled in
experimental systems. Gilliland et al have shown that BCR-ABL
alone can induce CML. But if murine bone marrow cells are
infected with a combination of viruses, one encoding BCR-ABL
and the other carrying an altered transcription factor, AML is
immediately observed in the transplanted mice.16
Gleevec is highly effective in chronic phase, perhaps because
the chronic phase involves only a single hit. However, in blast
crisis patients, Gleevec results in initial responses in virtually all
patients, but a rapid relapse and resistance follows. Much of this
resistance is due to mutations in the target BCR-ABL kinase12,17
indicating that the BCR-ABL remains an important target in this
disease. Perhaps other drugs can be identified to target the
mutant Gleevec-resistant forms of the BCR-ABL oncoprotein. It
is also likely that there are other pathways activated in blast
crisis. Therefore, the ultimate treatment will need target-directed
agents against both BCR-ABL and the other activated pathways.
Identification of these addition targets is crucial for complete
CML therapy.
Studies in AML have indicated the recurrent activation of or
alteration of various transcription factors. The first and bestcharacterized translocation was the t(15;17) translocation in
acute promyelocytic leukemia (APL), which juxtaposes the PML
gene onto the retinoic acid receptor alpha (RARa) subunit
gene.18–20 The most commonly targeted transcription factors in
AML translocations include the RAR and the CBF, which has as
its central DNA-binding component AML1 or RUNX-1, as well
as various homeobox genes.21
The activity of RAR is a paradigm for the function of altered
transcription factors in AML. The RAR acts as a dimer in concert
with a partner, RXR. It sits on the transcriptional control region
of a gene and acts to recruit a repressor complex.22–24 That
repressor complex includes nuclear co-repressor proteins and
principally the histone deacetylase complex (HDAC) that is
responsible for modulating chromatin, leading to gene silencing.22–24
This repression can be overcome by the addition of
retinoic acid (RA), which releases the repressor complex,
and recruits an activator complex containing P300.25,26 RA
acts as a potent differentiation agent in various hematopoietic
cell types.27
The PML-RARa fusion protein encoded by the (15;17)
translocation in APL binds the promoter and acts as DN
inhibitors of transcription through stable transcriptional repression.28 The repressor complex binds to the promoter, blocking
gene transcription and blocking expression of key genes
involved in hematopoietic cell differentiation. This repressor
complex is refractory to normal physiologic doses of RA.
Pharmacologic doses of RA release the repressor complex,
recruit the co-activator complex and restore the differentiation
of blood cells.29 A diagram of the PML-RARa complex and the
effects on gene transcription by RA and histone deacetylases is
presented in Figure 4.
This admittedly oversimplified model has nonetheless stimulated the idea that a target for this entire class of translocations is
the transcriptional repressor complex. The most attractive target
of this complex is the HDAC enzyme. Pharmacologic inhibition
of HDAC has been shown to relieve the repressor complex,
recruit the co-activator and restore hematopoietic differentiation.30,31
Recently, many HDAC inhibitors have been developed with
micromolar potencies and introduced into clinical trials.32,33 In
the laboratory, these have been shown to induce differentiation
of a variety of hematopoietic cells, and there is encouraging
clinical data in a growing variety of solid tumors as well as
leukemias.
Another emerging leukemia target is FLT3.34 FLT3 is a type 3/
class 3-receptor tyrosine kinase receptor that binds the growth
factor FLT3 ligand, an important cytokine for the maintenance of
hematopoietic stem cells. Naoe and colleagues have determined that about a quarter of AML patients has an internal
tandem duplication of a juxtamembrane region of the receptor,
which leads to its constitutive activation.35–38 Moreover,
another cohort of AML patients harbor point mutations that
involve the activation loop, a critical regulatory region around
the enzymatic cleft.39 Therefore, almost a third of all patients
with AML carry mutations in the FLT3 receptor in their leukemia
cells.
A number of FLT3 inhibitors are being evaluated in clinical
trails that have previously been shown to be active against
transformed cell lines and in mouse models.40,41 The clinical
activity of these agents is among the most exciting new
observations in target-directed treatment of AML.
Another excellent pathway for targeting in leukemia is the Ras
pathway. Ras is one of the canonical genetic lesions that is
responsible for driving leukemia and solid tumor cell proliferation.42 The Ras pathway can be activated at many levels.
Indeed, the FLT3 activating mutations themselves as well as
essentially all mutations in tyrosine kinases are upstream of the
Ras pathway, driving its activation and resulting in cell growth.
A diagram of activation of the Ras pathway in leukemia is
presented in Figure 5.
For example, BCR-ABL can associate with adaptor proteins
like Shc and Grb2 and activate Ras. Direct modulators of Ras
activity, such as the Ras GAP proteins, can be mutated or
activated in juvenile CML.43 There are many cases of
myelodysplasia in AML, which have direct mutational activation
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RA Larson et al
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Figure 4
PML-RARa translocation in AML. (a) In a normal cell in the absence of RA, a repressor complex is present on certain genes that may
modulate differentiation. (b) RA can induce a disassociation of transcription complexes on certain genes that may be involved in differentiation. (c)
In cells containing PML-RARa chromosomal translocation, differentiation is inhibited as it acts as a DN transcription factor and displaces RXRa and
secures HDAC.
Figure 5
Activation of the RAS pathway in leukemia.
of Ras. The Ras pathway can be activated in many ways and is
thus an excellent therapeutic target.
The enzymatic activity of Ras itself is not a particularly good
target, but Ras is activated in an obligate way by a posttranslational isoprenylation.44 A fatty acid moiety is added to the
C terminus of Ras by farnesyl transferase (FT). Ras is thereby
targeted to cell membranes, and a number of companies have
developed inhibitors of the FT that leave Ras in the cytosol as an
inactive enzyme.45–47
BCR-ABL is known to activate the Ras pathway. Dr Daley’s
laboratory has investigated whether farnesyl transferase inhibitors (FTIs) might serve as blockers of CML. In two different
mouse models, his laboratory has shown that treatment of mice
injected with BCR-ABL-transformed hematopoietic cells with
Ras inhibitors led to enhanced survival.48 Moreover, in a BCRLeukemia
ABL transgenic mouse model, the mice treated with FTIs had
prolonged survival compared to vehicle-alone-treated mice.49
FTIs also have activity in hematopoietic colony-forming
assays performed on cells taken from patient biopsies. BCRABL-positive colonies have a greater sensitivity to the drug than
colonies from normal individuals.48 These results suggest there
is therapeutic selectivity with FTI compounds, which could be
useful in the treatment of CML.
Together with Drs Mahon and Melo’s groups (Department of
Haematology, Imperial College of Science, Technology and
Medicine, Hammersmith Hospital, London, UK), Dr Daley has
shown that FTIs are also effective against cells from patients who
are resistant to Gleevec.50 Hematopoietic colony-forming assays
indicate that treatment with one micromolar Gleevec leads to
very low toxicity of BCR-ABL-positive cells from resistant
Novel approaches to treat leukemia
RA Larson et al
2363
patients. However, addition of an FTI at clinically achievable
levels generates complete suppression, suggesting that resistance to Gleevec does not imply cross-resistance to FTI. It is
therefore possible that FTI could be used successfully in
combination with Gleevec in CML patients.
Whether Ras is the true target of FTIs remains controversial.
Ras is one of several hundred proteins in the cell that require
post-translational modification by isoprenylation. There is now a
large body of literature implicating various other targets within
the cell that might be clinically relevant targets of FTIs.51–54
FTIs have been tested in early-stage clinical trials. Karp and
colleagues at the University of Maryland Greenebaum Cancer
Center (Baltimore, MD) have shown that the compound
Zarnestras, which has an oral route of delivery, was effective
in approximately 33% of the patients with highly refractory,
drug-resistant AML.55 There were also some CML blast crisis
patients in this study who had partial responses (PRs). Another
FTI, lonafarnib, has also been tested in early phase clinical trials.
Approximately 20–30% of patients had some clinical response
with this compound. There has been some modest compassionate use of this compound together with Gleevec in some
resistant patients. Combination clinical trials are being planned.
A summary of where the several oncoproteins that have been
discussed function in signal transduction pathway and where
various inhibitors act is presented in Figure 6.
In summary, understanding the total number of mutations in a
cancer cell will provide insight into which target-directed agents
are optimal for treating various malignancies. The CML-like
syndromes appear to be the result of single genetic abnormalities and therefore target-directed agents are likely to have
significant activity when used as monotherapy for these
disorders.
In contrast, the more acute malignancies, like acute leukemia,
typified by CML in blast crisis, arise from at least two genetic
lesions and perhaps more. Against acute leukemia, targetdirected agents are likely to be needed in combination. Dr
Gilliland has developed a very nice heuristic model to promote
this hypothesis. He classifies mutational lesions in AML as
falling into two classes. Class I mutations, which typically drive
cell proliferation, create a CML-like or myeloproliferative
condition. These have been mimicked in mice by expression
Figure 6
of BCR-ABL, TEL-PDGFR, and various activating mutations of
FLT3 and the Ras pathway.
Class I mutations exist in AML in concert with class II
mutations, which typically lead to loss of function of hematopoietic transcription factors, perhaps through stable recruitment
of HDAC or transcriptionally repressive complexes. Class II
mutations act to block differentiation, and when seen alone in
experimental models, induce an MDS-like condition.
How many drugs will it take to treat malignancies that harbor
multiple genetic mutations? If patients with the CML-like
syndrome are treated early post diagnosis with targeted kinase
inhibitors or perhaps the FTIs, significant single-agent activity
can be observed. However, in the more genetically complex
acute leukemias, combinations of target-directed agents will be
needed for effective therapy.
There is an enormous amount of enthusiasm because of the
tremendous success of Gleevec. However, this enthusiasm
should be tempered by the enormous challenges for targeted
therapy that remain. It is daunting to consider the task of
inhibiting multiple targets with highly specific agents. Each of
these drugs will have to be put through the very complicated
series of trials that are required before FDA approval. We are at
the beginning of a very exciting era of target-directed leukemia
therapy and once again, hematology seems to be paving the way
for all of oncology in understanding how to translate molecular
mechanisms into better therapies.
Questions from audience
Q: My question may be futile in this therapeutic new area, but
how do you reconcile the one-hit basis of CML with Dr
Fialkow’s data?
George Daley, MD: The question is that there are classical
data from Dr Fialkow and colleagues using clonality analysis
that suggested in some patients that there might be a pre-existing
clonal phase of hematopoiesis onto which the BCR-ABL
translocation is superimposed.56
These data suggest that there may be pre-existing genetic
lesions that contribute to the disease. I interpret the Fialkow data
to suggest that a pre-existing tendency toward clonal expansion
Site of action of oncogenes and signal transduction pathway inhibitors (black circles).
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RA Larson et al
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may indeed predispose individuals to the BCR-ABL translocation. CML is age-dependent and it tends to hit older rather than
the younger individuals. So perhaps clonality represents a
general precondition of the marrow, perhaps through decay of
telomeres, which generates increased frequency of chromosomal translocations. It is by no means clear, however, that the
Fialkow data imply a necessary prior genetic event.
We know in model systems, at least in the mouse, that the
single hit is functional. Emerging data from primary human cells
generated by transferring BCR-ABL into primary human tissue
indicate that a myeloproliferative phenotype can be seen in
primary CD34 þ cells. Dr Fialkow’s data and the experimental
data remain to be fully reconciled.
Q: You covered most of the genes, which are involved in
translocations. However, the ‘bad’ prognosis AML, for example,
partial deletions of chromosomes 5 and 7, cannot be cured with
chemotherapy. Even after bone marrow transplantation, the
outcome is poor. What is known about critical genes that are
involved in 5 and 7 chromosomal deletions?
George Daley, MD: This is indeed a good point. Chromosomal deletion syndromes are frequently seen in myelodysplasias
and secondary AMLs,57 and imply loss of tumor suppressor gene
function. A general principle I also did not touch on is gene
silencing – not just through transcriptional repression, but also
through methylation.58 This leads to the therapeutic concept of
demethylation agents, which may act to derepress or reactivate
some of these classes of tumor suppressor genes.
Q: Using very sensitive PCR, it is possible to demonstrate
many of these translocations in totally normal individuals who
never get leukemia. If one hit is enough, what is happening?
George Daley, MD: It is actually quite alarming when one
takes a sample of their own blood, and performs a sensitive PCR
and finds BCR-ABL, which some in my lab have done. The
reality is that the genetic lesion needs to occur in the right cell.
The presumption is that as the hematopoietic stem cell undergoes dramatic proliferative expansion through all the various
transit-amplifying populations, many translocations indeed
occur. However, most of the target cells are unable to sustain
the leukemia, because they are fated to ultimately differentiate,
apoptose or die. So at least two bits of bad luck are needed for
malignancy: the translocation has to occur in the first place and
it has to occur in a cell that can sustain a long-term leukemia,
like the hematopoietic stem cell.
of eight or nine transmembrane protein pumps that contain
ABCs.60–62 This is an energy-dependent process by which drugs
that passively cross the cell membrane are very quickly pumped
out. A number of these proteins have been characterized and
may be clinically relevant in patients with AML. This meeting
report will focus predominantly on MDR-1 or P-gp, because
most of the in vitro and clinical work has been carried out with
inhibitors of this particular pump. Many of the drugs used in the
treatment of AML are substrates for MDR-1 and are rapidly
extruded from drug-resistant cells.62,63 A diagram of the drug
pumps and the effects of pump modulators is presented in
Figure 7.
Multidrug resistance in AML (Charles A Schiffer, MD)
Drug resistance represents the major barrier to the successful
treatment of leukemia, and AML in particular. Why is AML so
resistant? First, AML is a disorder of hematopoietic precursors,
which are designed to ‘live forever’ until failure occurs in other
organ systems, such as the heart, kidneys or lungs. Excretory
organs are in constant contact with multiple types of toxins,
particularly from the organisms that we live with, and have
developed mechanisms to survive such exposure. For example,
most resistance mechanisms are amplified in gastrointestinal
(GI) tract precursors. Similarly, bone marrow purging experiments indicate that hematopoietic stem cells can survive
exposure in vitro to large doses of a variety of chemotherapeutic
agents.59 Therefore, it is not particularly surprising that
leukemias that derive from cells that resemble hematopoietic
stem cells (eg, Ph-positive diseases, myelodysplasia and all the
myeloproliferative diseases) are very resistant to chemotherapy.
A major cause of resistance is a consequence of active efflux
of drugs that have entered the cells, usually mediated by a group
Leukemia
Figure 7
Effects of drug pumps and modulators on chemotherapeutic drug resistance.
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RA Larson et al
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Overexpression of P-gp can be detected in AML blasts at
diagnosis, particularly in older patients, and may be further
increased at relapse.63–67 In many studies, P-gp overexpression
is associated with a poorer outcome, as is overexpression of
some of the other efflux pumps, although the data on P-gp seem
to be the strongest in this regard.64,66 Importantly, P-gpmediated resistance can be reversed in vitro by a variety of
drugs, such as cyclosporine, PSC-833 and quinine.63,66 These in
vitro observations led to a series of clinical trials evaluating
modulators of drug resistance in patients with AML.
A complicating issue is that the administration of some of the
MDR1 inhibitors affects the metabolism and disposition of many
chemotherapeutic drugs because of their effect on normal
tissues such as biliary lining cells. In general, when modulators
of these efflux pumps are given to patients, there is impaired
excretion of the chemotherapeutic drug, increases in AUC and
drug exposure and the potential for increased toxicity.
In vitro assays of P-gp
When in vitro assays on hematopoietic cells from AML patients
are performed, they are usually performed on whole populations
of leukemia cells. A problem, however, is that these cells are
quite heterogeneous. For example, within a population of
leukemia cells with similar morphology, there are dramatic
differences in expression of different surface antigens. Furthermore, only a small fraction of the leukemia cells have the
capacity to grow in long-term culture or in SCID mouse models.
This subpopulation has been characterized by cell sorting as
CD34 þ , HLA and DR negative. Animal experiments have
shown that injection of this subpopulation of cells is capable of
producing sustained tumors.68 Experiments with blasts from
patients with AML have indicated that o1% of cells had the
potential to produce long-term persistence either in culture or in
animals, and might therefore represent leukemic ‘stem cells’
P-gp expression can be measured with antibodies directed
against membrane P-gp epitopes by flow cytometric analysis or
by measuring the function of the pump.69 Rhodamine 123 dye
can be used to measure drug pump function as a surrogate for
chemotherapeutic drugs. In ‘normals’, incubation with rhodamine results in increased fluorescence. In resistant cells
however, there is decreased fluorescence because of rapid
excretion of the dye. The effect of different modulators or pump
inhibitors can also be evaluated in such assays.
It can be difficult to detect drug resistance in a heterogeneous
hematopoietic cell population. If only 0.5–1% of the cell
population are actually responsible for persistence of leukemia
after treatment, even very sensitive flow cytometric assays might
be incapable of assessing the characteristics of this very small
subpopulation of cells that may be most important in terms of
drug resistance. To place this point in clinical perspective, even
marginally effective drugs produce a multilog reduction in the
number of AML cells that are circulating or in the bone marrow.
Thus, it is the very small subpopulation of residual cells that are
responsible for ultimate drug resistance and the patient’s death.
Interpretation of in vitro assays of MDR can also be difficult. It
has been known for years that there is variable and sometimes
poor concordance among the immune, molecular and functional assays used to assess the effects of P-gp expression. Most
laboratories now stress the importance of functional assays
measuring the efflux/retention of the particular drug of interest. It
is inferred in flow cytometry experiments that increased
immunofluorescence representing increased chemotherapeutic
drug retention results in increased cytotoxicity. However, a
clinically relevant definition of ‘positive’ in any of these assays is
not well defined and the magnitude of the in vitro fluorescence
necessary to confer drug resistance in the patient is not known.
Moreover, it is not clear which cells are killed – the clonogenic
or the bystander cells. Lastly, it is not clear that the behavior of
rhodamine, one of the substrates commonly used in drug efflux
experiments, is similar to the effects of clinically important drugs
such as the anthracyclines. It will take cumbersome clinical
trials to answer these questions, which can take a long time and
often can be difficult to analyze.
Clinical trials with MDR modulators
A number of clinical trials evaluating MDR modulators in AML
have recently been completed. The CALGB completed a Phase I
study, which was a prelude to a planned Phase III study, in older
patients with previously untreated de novo AML.70 Patients
received combination therapy with daunorubicin, etoposide and
cytarabine with or without the MDR-1 modulator, PSC-833.
The maximally tolerated dose without the modulator was
60 mg/m2 3 doses of daunorubicin, 100 mg/m2 3 doses of
etoposide and 100 mg/m2 of cytarabine for 7 days. With the
addition of PSC-833, however, the MTD were approximately 1/
3 lower (40 mg/m2 of daunorubicin and 60 mg/m2 of etoposide)
with mucositis as the dose-limiting toxicity. This was one of the
largest Phase I trials that has ever been performed in acute
leukemia, with results consistent with other studies demonstrating the necessity of dose reduction because of the effect of the
MDR inhibitor on the pharmacokinetics (PK) of the chemotherapeutic agents. Trials using such modulators with reduced doses
of chemotherapy are based on the hope that intracellular drug
levels and resultant cytoxicity of the clonogenic cells will be
increased to a much greater extent than can be achieved with
higher doses alone. Indeed, earlier clinical trials suggest that
higher doses of etoposide and/or daunorubicin are not sufficient
to improve the outcome of treatment with AML.
CALGB 9720 was an extension of the Phase I study in which
older people with de novo AML were randomized to receive the
two regimens described above.71 Unfortunately, this study was
closed early because of excessive toxicity and mortality in the
group receiving PSC-833. After about 120 patients were
analyzed, there was a somewhat lower complete response rate
in the experimental group, but a much higher early death rate
associated with mucositis and infection. These results could not
be explained by differences in the clinical characteristics in the
two groups of patients. Overall, MDR-1 expression seemed to be
a prognostic factor, in that the CR rate was higher in patients
who were efflux-negative.
The ECOG did a study evaluating PSC-833, which was also
stopped early because of poor results. This study enrolled very
high-risk patients including those in early relapse, with primary
refractory disease, high-risk MDS or secondary leukemias and
others who were post marrow transplantation. The patients were
treated with a regimen of mitoxantrone, etoposide and ara-C
with or without PSC-833.72,73 The doses of mitoxantrone and
etoposide were reduced in the PSC-833 cohort. There was no
difference in overall or disease-free survival, but because of a
significant decrease in the CR rate in the PSC-833 arm, this study
was stopped prematurely.
In addition to the CALGB and ECOG studies, another large
randomized trial conducted in Europe has been completed.
While this study has not yet been published, it also failed to
show an advantage of adding PSC-833 to induction therapy for
older patients with AML.
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Somewhat in contrast are the results of a study performed by
SWOG using cyclosporine as an MDR modulator.73 PSC 833 is
a nonimmunosuppressive, non-nephrotoxic analog of cyclosporine, which was much more active in vitro and hence was
preferred by some groups in clinical trials. The SWOG study
included high-risk patients with AML who received cytarabine
and daunorubicin with or without cyclosporine. The same dose
of daunorubicin was used in each arm, although a potentially
important difference was that the daunorubicin was administered by continuous infusion rather than by the standard bolus
used in the CALGB and ECOG studies. Responders received a
second course of the same regimen. More than 200 patients
were randomized, of whom more than half were in first relapse
and almost 15–20% were primarily refractory. Among the
patients who achieved remission, the relapse-free survival was
significantly higher in the group of patients who received
cyclosporine compared to the control group and what would
have been predicted from historical experience. This translated
into an overall survival advantage from the time of randomization for the cyclosporine recipients.
What can be learned from these trials that can be applied in
future studies and why have attempts to modulate P-gp in AML
been disappointing? An obvious issue is that there are additional
mechanisms of resistance not affected by PSC-833.73 An
intriguing possibility is that cyclosporine might have an effect
on these other resistance factors accounting for the positive
results in the SWOG trial. There are also competing causes of
treatment failure in AML, which present a design and statistical
conundrum. At least a third of older AML patients will die of
complications of treatment, which have nothing to do with
whether MDR is successfully modulated. Although all patients
should be included in the analysis of randomized trials, a high
fraction of patients with early death dilute the ability to detect a
biologic effect of a modulator of MDR.
Furthermore, it is not known whether the modulator, even in
vitro, enhances the killing efficiency of the critical clonogenic
leukemia cells. Although it is possible to achieve levels in vivo
that are effective in vitro, it is not known whether this occurs in
every patient since there is considerable PK variability among
individuals and higher levels may be needed in vivo to reverse
drug resistance effectively. Another possibility is inadequate
exposure to chemotherapy in the patients who are receiving
reduced doses of chemotherapy with the expectation that the
modulator will actually increase the drug exposure. If this PK
effect does not occur reliably in every patient, it is possible that
some individuals are actually being underdosed with chemotherapy with the potential for inferior outcome.
AML may also not be the ideal tumor to evaluate the concept
of MDR modulation. Only complete response is clinically
relevant in AML, presenting a significant challenge for any new
therapy. In contrast, in other diseases such as multiple myeloma
or in certain solid tumors, partial remission is clinically very
meaningful and may be more readily achievable. In addition,
early death is not a major issue in such tumors.
Lastly, clinical trials have focused on high-risk patients
because there are lots of them and if a true benefit is observed,
it could be very obvious and detected more rapidly than in
patients with a better prognosis. However, these are also the
hardest AML patients to treat, and small but clinically important
differences could be missed particularly since such trials include
a biologically heterogeneous population. There is also some
disagreement about what the relevant end point should be.
Obviously overall survival is critical, but some researchers have
focused on the CR rate, or the fraction of patients who are cured
rather than median survival. These issues have to be considered
Leukemia
when clinical trials are designed and affect sample size and
study feasibility.
Other MDR modulators
There are a number of other drugs that affect MDR, which are in
early clinical trials. These include Zosuquidar.HCl trihydrochloride (LY335979), which is reported not to have a PK
effect on anthracycline disposition.74,75 Phase I trials in
combination with standard dose chemotherapy have been
completed and ECOG is contemplating a Phase III trial in
AML patients utilizing this drug.
Other compounds include VX710, which has the potential
advantage of inhibiting MRP1 but which also had a significant
effect on paclitaxel disposition and was associated with
hyperbilirubinemia suggesting an effect on hepatic function.76,77
KXR9576 is an anthranilic acid derivative, which can be
administered orally and intravenously.78,79 It has a prolonged
effect after in vitro exposure to cells and it is presently in Phase I
and II trials. In these studies, natural killer (NK) cells were
isolated and characterized ex vivo after XR9576 infusion. NK
cells normally overexpress MDR and in some patients, there was
marked inhibition of rhodamine efflux after infusion of this MDR
inhibitor, demonstrating a significant in vivo effect.
R101933 is an MDR inhibitor that can be administered orally
and intravenously. Side effects have included somnolence; no
PK effect was demonstrated when used in combination with
docetaxel.80 Lastly, GG120918 may have more pleiotropic
effects in that it also modulates the BCRP.66
Future considerations
What is next in drug resistance studies in AML? As noted, there
are modulators with less PK effects, which are of considerable
interest. It might be relevant to focus only on newly diagnosed
younger patients, in whom an additional goal could be the
prevention of the emergence of resistance. The CALGB has
completed such a Phase I trial in younger patients with PSC-833
and has a randomized study in younger patients in progress. Pgp is also overexpressed in patients with acute lymphocytic
leukemia, and there is enormous room for improvement in the
treatment of adult ALL. Given the multiagent regimens used in
ALL, however, ‘ideal’ trials with MDR modulators will be
difficult to design.
Finally, although a lot is known about drug resistance, little is
known as to what promotes apparent drug sensitivity. For
example, even in the poorest prognosis leukemias, a fraction of
patients are cured with chemotherapy alone. The blasts from
patients with acute progranulocytic leukemia, which are
exquisitely sensitive to the effects of anthracyclines, have low
expression of MDR1. Certain transcription factor mutations, TEL,
AML1, AML1-ETO, can actually repress MDR expression by
interacting with the promoter region of the MDR.81,82 These turn
out to be highly curable leukemias. In other patients, differences
in the cytokinetic state of the leukemic stem cell or immunemediated suppression of minimal residual disease may contribute to disease cure. It is hoped that greater understanding of
these mechanisms of drug sensitivity and resistance will
translate into improved outcomes in the future.
Questions from audience
Q: I just wanted to make a comment about biricodar. I should
point out in the interest of fairness that I am working with the
Novel approaches to treat leukemia
RA Larson et al
2367
company that has tried to develop that drug. Your point about
elevations in bilirubin is well taken; certainly if this occurs,
it could affect the PK of anthracyclines. However, those
elevations were very modest, and when one actually looks at
very carefully done studies with PK of anthracycline alone and
then in combination with biricodar, there are no substantial
differences in the PK. These studies have also been extended to
other types of drugs, such as mitoxantrone, and the results seem
fairly consistent. So while I share your concerns, particularly in
this very delicate population of patients, the results are
encouraging.
I have another point and I ask your thoughts about this.
There have been a lot of questions raised about the
cyclosporine results. I certainly view them as extremely
encouraging. Is it conceivable that any of these results could
occur by other effects of cyclosporine? I am curious about
your views on this or do you think the data are done carefully
enough to suggest that this occurs primarily by an MDR
mechanism?
Charles Schiffer, MD: With regard to the first comments, I
thank you for those. I could not find literature or publications
that showed that, but if indeed there is no effect on PK,
particularly of the leukemia-relevant drugs, that is very
important. I think it is easier to do a randomized study where
you do not have to drop the dose of your chemotherapy in the
experimental arm.
Cyclosporine is a little more pleiotropic than we thought
in terms of inhibition of other ABC pumps and that could
be one explanation for the SWOG results, because it clearly
had some benefit in that trial with patients who were not
MDR-positive.
Q: I have a question regarding secondary neoplasias, maybe
as a consequence of MDR1 inhibition. You mentioned in the
beginning of your talk the essential meaning of MDR1 in
physiologic tissues. What about late consequences of MDR1
inhibition when anthracyclines or other toxic drugs are being
administered?
Charles Schiffer, MD: I must say, I have never thought about
it. I have always assumed that these patients would be happy to
have had that opportunity. In addition, the period of exposure is
very brief. I guess, however, one could hypothesize that if you
had an early intestinal lesion where MDR1 is an important
component in the GI tract stem cell, that you might protect it in
some way I am not aware of human or any animal data, but it is
an interesting thought.
Q: My question concerns the issue of stem cells and how
important MDR is to the basic stem cells. When one examines
the negative data – not the stuff by List, but the ECOG things you
showed – is it lack of effect of the drug in PSC or is it the
increased toxicity of the combination that results in the negative
trial? I mean, is MDR so important in the basic stem cell that it is
where the problem lies?
Charles Schiffer, MD: I think it is both. With regard to the first
question, there is increased toxicity and some increased
mortality, and that becomes particularly evident if more fragile
patients are examined. For example, in the CALGB study, in
younger patients the toxicity was less of an issue. So I think it is a
function of both.
Recall that a positive trial in this disease is often obtained if
the outcomes are improved by 10%. Oncologists would be
ecstatic if that happened. However, with all the variables I have
listed, one could see how even in large trials that have a couple
of hundred patients, small differences could be obscured. This is
why this has really been a very difficult field to study and to
perform clean clinical trials.
The evolving role of monoclonal antibodies in the treatment
of CLL (Pierluigi Porcu, MD)
CLL is the most common type of adult leukemia in the Western
world.83 Most of the patients who develop CLL will need
treatment at some point during the course of the disease, and
many eventually will die from complications related to the
disease or its treatment. CLL is not curable with conventional
treatments. For a long time, CLL was considered a relatively
simple and even unexciting form of leukemia, compared to the
acute leukemias. This was more a reflection of the simplicity
and limitations of the treatments available rather than lack of
biological or clinical complexity. The complexity of CLL was
initially recognized with the staging classifications of Rai and
Binet. Greater therapeutic sophistication in CLL was first
introduced with the independent discovery by Dr Grever (Ohio
State University, Columbus, OH, USA) and Dr Keating (MD
Anderson Cancer Center, Houston, TX, USA) that the purine
analog fludarabine was able to induce significant responses in
patients with refractory disease.84,85 Eventually, fludarabine was
taken to front-line therapy with impressive results86 and is now
being used alone or in combination with alkylating agents and
monoclonal antibodies, producing a high number of complete
responses.
Drs Rai (Division of Hematology/Oncology, Long Island
Jewish Medical Center, New Hyde Park, NY, USA) and Binet
(Laboratory of Hematology, CHRU Bretonneau, Tours, France)
recognized the existence of different prognostic groups of CLL
patients.87–89 Even though the classifications are slightly
different, both recognized that three major prognostic groups
were with different survivals. More recently, advances in our
knowledge of the molecular biology of CLL cells have led to the
recognition of new biological prognostic markers, which can be
integrated into the classifications of Drs Rai and Binet. Intriguing
data about mutations in the variable region of the heavy
immunoglobulin (Ig) chain (IgVH) in CLL cells have recently
been reported.90,91 It is now recognized that there are two forms
of CLL, one where the IgVH region contains somatic hypermutations (SHM), indicating that these cells are derived from
germinal center (GC) B cells. These patients have a much better
prognosis compared to patients with the second form of CLL,
who do not have SHM in the IgVH region. A second type of
biological risk stratification relies on the detection of specific
chromosomal translocations in CLL cells.92–94 Three different
subgroups of patients have been recognized who have
remarkably different lengths of survival and treatment-free
intervals (TFI), depending on which chromosomal aberration
they carry. The presence of 17p deletion or 11q deletion are
associated with clinically more aggressive disease and worse
prognosis; patients with normal karyotype or trisomy 12 have an
intermediate-risk prognosis; finally, patients with 13q deletion
have a better outcome.
A class of compounds that must be considered among
targeted therapies is that of mAbs. They can be designed to
target specific molecules important for the biology CLL cells,
such as survival, proliferation and trafficking. A list of chimeric
and humanized mAbs that are approved or in clinical
development for the treatment of CLL is presented in Figure 8.
Monoclonal antibodies have become a new paradigm in the
treatment of hematologic malignancies.95,96 First, the toxicity is
non-overlapping with that of chemotherapy and generally
myelosuppression is not observed. They also have excellent
biodistribution in key anatomical areas, such as the bone
marrow, peripheral blood and spleen, that are critically involved
in CLL and lymphoma. They have a novel, albeit incompletely
Leukemia
Novel approaches to treat leukemia
RA Larson et al
2368
Figure 8
Use of engineered antibodies to treat CLL.
understood, mechanism of action. Finally they have very broad
spectrum of targets, such as surface antigens, cytokines,
adhesion molecules, etc.
One of the best-known monoclonal antibodies is rituximab
(Rituxans), a chimeric antibody, which binds to the CD20
molecule expressed on B cell. It has significant single-agent
activity in low-grade B-cell lymphoproliferative disorders and
modest single-agent activity in diffuse large-cell lymphomas,
with a very favorable toxicity profile. In CLL, early studies in
previously treated patients using the standard weekly 4
schedule did not show a significant activity, but when
previously untreated patients were examined it was recognized
that standard-dose rituximab had reasonable activity.97 However, it is clear that, comparatively, standard-dose rituximab in
CLL and small lymphocytic lymphoma (SLL) is substantially less
active than in low-grade lymphomas.
There may be different reasons for this phenomenon. One is
the different PK of rituximab in CLL and SLL as opposed to lowgrade lymphomas. Other possibilities include a deficient or
different mechanism of mAb-mediated killing compared to lowgrade lymphomas, resistance to complement-mediated killing
and decreased CD20 density in CLL cells. Additionally, there
may be a deficiency in immune-effector cells in CLL. These
patients have significant underlying immunodeficiency related
to their disease and they then are treated with alkylating agents,
steroids and fludarabine, which add to the immunosuppression.
Finally, defective signaling events leading to reduced sensitivity
to apoptosis-inducing signals may also play a role.
The problem of unfavorable PK has been addressed clinically
in two different ways. O’Brien et al (Leukemia Department, The
University of Texas MD Anderson Cancer Center, Houston, TX,
USA) performed a dose-escalating trial with rituximab, which
was taken up to 2.25 g/m2 as a weekly dose.98 This study
showed that dose escalation could be accomplished safely, and
that the response rates were increasing with higher doses of
rituximab. A second approach was adopted by Byrd et al
(Division of Hematology-Oncology, Ohio State University,
Columbus, OH, USA), which used a three times a week
administration of rituximab.99 This led to substantially higher
Leukemia
plasma levels of rituximab and also was associated with
significant response rates in previously treated patients. These
two studies illustrate how part of the resistance of CLL cells to
rituximab might be overcome with higher plasma levels of
antibody.
Several questions remain, however, as to the specific
mechanism of rituximab’s action in CLL as opposed to lowgrade lymphoma. One of the currently favored mechanisms of
action of rituximab in B-cell malignancies is that it works
through antibody-dependent cell cytotoxicity (ADCC). A significant amount of evidence suggests that ADCC is very
important for the activity of rituximab. However, many of the
antibodies that are currently used in the treatment of cancer are
also known to induce intracellular signaling when tumor cells
are examined in vitro, and the possibility that rituximab-induced
signaling plays a role in its antitumor activity in vivo should be
carefully examined.
ADCC is mediated by a set of cell surface receptors that bind
the Fc portion of the immunoglobulin G (FcgR). While many
isoforms of FcgR exist, they can generally be distinguished as
activating or inhibitory,100 depending on the effect that their
engagement has on the ability of the effector cell to perform
ADCC. Many innate immune effector cells carrying these
receptors can efficiently mediate ADCC, remarkably among
them NK cells, which only express the activating receptors.101 In
contrast, other cells such as B cells and monocytes express some
inhibitory receptor that can turn off ADCC.
Jeffrey Ravetch’s group (Laboratory of Molecular Genetics and
Immunology, Rockefeller University, New York, NY, USA), has
very elegantly demonstrated the importance of FcgR in antibody-mediated tumor killing in vivo.102 They performed a study
with mice infused with B16 melanoma cells. In wild-type mice
given no treatment, massive pulmonary metastases were
observed. If antibodies against the B16 melanoma cells were
administered to the wild-type mice, a reduction of the incidence
of pulmonary metastases was observed, although a substantial
amount of disease was still present. However, when knockout
mice for the inhibitory form, but not the activating form, of the
FcgR were administered the antibody, there was a near-
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RA Larson et al
2369
complete clearance of pulmonary metastases, indicating
that in vivo the presence of inhibitory FcgR was decreasing the
efficacy of the antimelanoma monoclonal antibodies.
These experiments strongly suggest that in vivo, ADCC depends
on the balance between inhibitory and activating FcgR
and that alterations of this balance have profound effects on
the outcome of therapy with mAbs. A way to increase the
balance in favor of ADCC, with the goal of enhancing
the efficacy of mAb-based therapy, is to increase the
number of cells that are carrying the activating FcgR by giving
a cytokine such as interleukin-2 (IL-2) that expends NK cells.
Some of these approaches are being explored in lymphoma by
many different research groups.103 On the other hand, Th2
cytokines such as IL-4 and IL-10, which increase the expression
of inhibitory FcgR on these cells, are associated with a reduced
ADCC effect.
CLL, however, is a difficult disease target from the standpoint
of ADCC for a number of reasons. First, ADCC usually requires a
favorable effector-to-target ratio, which is very difficult to
achieve in CLL because of the high number of circulating tumor
cells. Second, patients with CLL tend to have a Th2 cytokine
profile104–106 that downmodulates ADCC. Third, CLL cells
express the inhibitory FcgR.107 Finally, experimental studies of
ADCC in CLL are difficult to perform because there is no good
preclinical model for human CLL at this time and it is hard to
isolate sufficient effector cells from the peripheral blood to
perform in vitro ADCC assays. Based on these considerations,
one would predict that ADCC may not play a major role in the
antitumor activity of rituximab in CLL.
Induction of apoptosis may be an alternative possibility for the
effect of rituximab in CLL.108 If CLL cells are incubated with
rituximab in vitro, particularly when crosslinking antibodies are
added, a substantial amount of apoptosis is observed. Evidence
for a role of induction of apoptosis also comes from studies
examining the signaling activity that follows rituximab binding
to CLL cells in vivo. The proform of caspase-3 is converted into
the active p16 form in cells taken from patients after a 1- to 3day exposure to rituximab.109 The level of antiapoptotic
molecules such as XIAP and MCL-1 following exposure to
rituximab is also reduced. Of note, caspase activation correlates
with reduction in lymphocyte counts following rituximab
therapy. A model for the mechanism of action of rituximab in
CLL has therefore been proposed.109 Exposure to rituximab
results in activation of the caspase pathway of apoptosis. Both
caspase-3 and caspase-9 are activated. This results in activation
of the downstream effectors of apoptosis. Additionally, two
antiapoptotic molecules, MCL-1 and XIAP, are down-modulated
in CLL cells following binding of the antibody, potentially
making the cells more sensitive to the action of other drugs
as well.
What has been learned from the single-agent rituximab
studies in CLL? First, rituximab is active as a single agent,
particularly when the dose and the schedule of administration
are targeted to reach a significant blood level of the antibody.
Second, rituximab is safe, and excessive myelosuppression or
infectious complications are not observed. Third, the apoptotic
pathways activated in response to the binding of rituximab to
CD20 are likely to have a major role in the antitumor activity of
this antibody in CLL.
How to improve on rituximab therapy? One obvious way is to
combine the antibody with other active agents, such as
fludarabine or alkylating agents. Another is to combine it with
agents that down-modulate known survival signals for CLL cells
coming from the microenvironment, among them tumor
necrosis factor-a (TNF-a).
To determine whether rituximab can be taken to the upfront
stage in combination with fludarabine, CALGB conducted a
clinical trial (CALGB 9712) where patients with newly
diagnosed CLL were randomized to receive either fludarabine
followed by rituximab or a concomitant regimen of fludarabine
and rituximab followed by maintenance rituximab.110 A total of
104 patients were involved. A good number of them had
intermediate risk and about half of them had high risk. The
response analysis was intriguing, particularly in terms of the
complete responses, indicating that there was a significant
difference between the concurrent and the sequential arms, with
47% and 28% response rates respectively. These results suggest
that rituximab was increasing the sensitivity of CLL cells to the
cytotoxic effect of fludarabine. The other important observation
in this trial was that this result was achieved at no cost of
increased toxicity. In terms of the incidence of opportunistic
infections, there were more opportunistic infections in the
sequential as opposed to the concurrent arms. In terms of
hematological and nonhematological toxicity, there was slightly
more, but not significant, hematological toxicity in the
concurrent arm.
Some of the correlative studies performed by Dr Byrd et al in
their initial trial of single-agent rituximab in CLL patients aimed
at understanding the mechanism of the severe infusional
reactions to rituximab, observed in CLL patients. Byrd et al
observed that patients who were having severe infusional
reactions had significantly higher levels of TNF-a as opposed
to those who did not. TNF-a appears to be associated not only
with toxicity, but it is also likely to be responsible for protecting
CLL cells from apoptosis. TNF-a signaling through its receptor
induces activation of NF-kB, which increases Bcl-2 transcription. This signal therefore not only increases survival, but also
blocks the proapoptotic signaling induced by rituximab binding
to CD20 in CLL cells. Therefore, TNF-a may become an
attractive target from the therapeutic standpoint in addition to
being a target in terms of reducing the infusional reactions. A
clinical trial examining the combination of rituximab and
etanercept, a dimeric fusion protein that binds TNF-a, blocking
its interaction with cell surface receptors,111,112 is in progress at
the Ohio State University. The hypothesis behind this trial is that
by reducing the availability of TNF-a to CLL cells, there will be
an increase in the efficacy of rituximab and potentially a
reduction in the incidence of severe infusion reactions.
Campath-1H is a humanized anti-CD52 antibody and has
significant activity in T-cell prolymphocytic leukemia (T-PLL),
CLL and cutaneous T-cell lymphomas (CTCL).113 The target
antigen, CD52, is a GPI-linked glycoprotein whose function is
not well known. CD52 is broadly expressed in B cells, T cells,
monocytes, NK cells and dendritic cells.115 The mechanism of
action of Campath-1H is not known. A clinical trial with
Campath-1H was conducted in fludarabine-refractory patients.114–116 Campath-1H was administered intravenously by
a stepped-up dosing with 3, 10 and 30 mg over a week and then
followed by about 12 weeks of three times a week administration, with PCP and varicella zoster prophylaxis. In this pivotal
trial, 92 patients were enrolled. The overall response rate was
33%. This was remarkable for fludarabine-refractory patients,
who before the availability of Campath-1H had no viable option
for therapy. The median survival was 16 months. Importantly,
among the people who actually responded, there was a
significant increase in median survival.
Toxicity was substantial, both at the hematological and
nonhematological level. A 50% incidence of grade 3/4
neutropenia and thrombocytopenia was observed. Infections,
fever, rigors, rashes, nausea and vomiting, which were difficult
Leukemia
Novel approaches to treat leukemia
RA Larson et al
2370
to manage, were also observed. Patients required antiemetics.
Some deaths occurred from infection. An additional complication was frequent reactivation of cytomegalovirus (CMV).
CALGB is now conducting two studies with Campath-1H.
One consists of fludarabine induction, followed by randomization to intravenous Campath-1H vs a new subcutaneous (SQ)
schedule of administration, which seems to be associated with a
significantly lower incidence of infusional reactions. A second
CLL trial in CALGB is CALGB10101, which is looking at the
fludarabine–cyclophosphamide–rituximab (FCR) regimen to be
followed by subcutaneous Campath-1H. In both of these trials,
the principal goal is to achieve the highest possible level of
complete remission in front-line patients with CLL.
A third mAb in clinical trials for CLL is Hu1D10.117 Hu1D10
targets an epitope on the HLA-DR beta chain, which is
expressed on many hematopoietic cells, including B cells,
monocytes, T cells and dendritic cells. Altogether, about 50–
60% of B-cell lymphomas and approximately 75% of CLL
patients express this epitope. Hu1D10 can mediate ADCC and
complement-dependent cytotoxicity (CDC), and induce apoptosis in vitro.118,119 Similar to rituximab, when Hu1D10 is
combined with crosslinking antibodies, the amount of cells that
undergo apoptosis is significantly higher. Unlike rituximab,
though, Hu1D10 does not activate caspase-3 or PARP cleavage.
Hu1D10 reduces the mitochondrial membrane potential in CLL
cells (JC Byrd, unpublished results), suggesting that the
mechanism of induction of apoptosis in CLL cells with
Hu1D10 might be different from that of rituximab.
A Phase I study in lymphomas was recently completed. There
were four drug levels with a standard weekly 4 schedule,
stepping up from 0.15 to 5 mg/kg. In one cohort, there was an
attempt to administer a more intensified treatment with daily
incremental doses of Hu1D10 over a week. Some intriguing
responses were observed in this Phase I trial in lymphomas,
which led to a Phase II trial in low-grade lymphoma and an
additional pilot Phase I trial in CLL and ALL. In the Phase II study
in low-grade lymphoma, preliminary data do not appear to
confirm the activity of this antibody. In CLL and in ALL, a Phase I
study was initiated in collaboration between the Ohio State
University and the University of Chicago. The schedule of
administration was thrice weekly. This schedule was adopted
because the antibody has a very short half-life. A stepped-up
dosing was chosen, similar to the one used for Campath-1H, to
decrease infusional toxicity. The study design was to gradually
increase the dose of Hu1D10 to reach the target dose level for
each particular cohort, which were 1.5, 3 and 5 mg/kg. In seven
of 11 evaluable patients, there was a significant decrease in
tumor volume at each dose level. Several patients had
stabilization of their disease. There was a PR in a patient with
a 17p deletion. This was very interesting, as these patients are
known to be insensitive to rituximab. Moderate toxicity was
observed with relatively unusual adverse events, such as
delayed infusion toxicity with hypotension even several hours
after the completion of the infusion, grade 3 hypophosphatemia,
rash and urticaria. There were some cases of thrombocytopenia.
Nausea was reported in these studies as well as headaches,
which occurred in a significant number of patients. Based on the
observation that Hu1D10-induced apoptosis in CLL cells
follows different signaling pathways compared to rituximab
(caspase-independent vs caspase-dependent, respectively) and
the preliminary evidence of clinical activity in CLL, further
development of this antibody in the treatment of CLL is being
pursued.
In conclusion, cytogenetics and IgVH mutational status are
new powerful tools for risk stratifying CLL patients at diagnosis
Leukemia
and for treatment. This is a significant advance in terms of the
planning of further clinical trials. The mAbs active in CLL may
work differently in CLL as compared to non-Hodgkin’s
lymphomas. Intriguing is the possibility that different mAbs
may eliminate specific subsets of CLL clones, depending on
which molecular abnormalities they carry. Therefore, combining different antibodies and biologic therapies should be the
focus of the next generation of clinical trials.
Questions from the audience
Q: One of the hazards that seems to have emerged is the
proximity of giving fludarabine followed by Campath-1H. A
recent German trial has shown prolonged cytopenias accompanied by very profound fungal infections. Would you like to
comment on that and how long a gap you really need to leave
between the two?
Pierluigi Porcu, MD: I would say that the approaches that are
being taken right now are to wait several weeks, 4–6 weeks. I
think that it also depends on the severity of myelosuppression
following fludarabine, which may be significantly different from
patient to patient. So, it is going to be difficult to have a
homogenous approach that will be effective for all the patients.
Q: I think the German trials are really quite worrying, because
they did not leave even as much as 2 months, but there are
several trials that are leaving 8 weeks. I am hesitant to say that is
going to be enough, because they were getting 6 months of
neutropenia after that combination.
Pierluigi Porcu, MD: Yes, I agree that the issue of hematological toxicity is extremely important.
Richard Larson, MD: I can comment on that, perhaps. In the
CALGB study, there were only four cycles of fludarabine
administered followed by a 2-month interval and then followed
by 4 weeks of Campath-1H therapy. Fungal infections were not
observed but reactivation of CMV occurred. Therefore, there is
some concern about the combined immunotoxicity of that
sequential regimen.
Q: I think that the major challenge that we are facing now is
when to treat early-stage A Binet CLL. I think that approximately
50% will never be treated and will live 20–30 years without any
complications. So do you have any idea of the place of these
antibodies in the treatment of CLL?
Pierluigi Porcu, MD: I think that most of the combination
approaches that are being explored now will have to be tested in
Stage A Binet CLL patients who have high-risk disease defined
by FISH or IgVH mutational status, as the toxicity of these
combinations remains a big concern. This is only a minority of
Binet A patients but one with no good expectations for a
prolonged treatment-free interval or survival. I think that for
patients who have good-risk early-stage CLL, deciding whether
or not they should go on one of the clinical trials with
combination of antibodies and fludarabine or antibodies,
fludarabine, and alkylating agents remains a great challenge.
A patient who might need therapy and is not willing to be
treated on one of those trials, or has concerns about toxicity,
could be treated with single-agent antibodies, since they are
substantially more active in newly diagnosed patients.
The remaining challenges in ALL (Ching-Hon Pui, MD)
As cure rates in pediatric ALL reach 80%, emphases are placed
on precise risk-directed therapy to avoid over- or undertreatment, to elucidate the mechanisms involved in drug
Novel approaches to treat leukemia
RA Larson et al
resistance and to develop new therapeutic strategies.120 Genetic
analyses have contributed greatly to our understanding of the
pathogenesis and prognosis of ALL, and have begun to identify
molecular targets for specific treatment.121 Heretofore, specific
genetic abnormalities with prognostic relevance were limited to
cases of B-cell precursor ALL. By use of DNA microarrays, Tom
Look and colleagues (Harvard University, Boston, MA, USA)
recently classified T-cell ALL into four major subtypes with
prognostic significance: MLL-ENL and HOX11 clusters are
associated with a favorable prognosis and TAL1 and LYL1
clusters with inferior outcome.122 Altogether, approximately
75% of childhood ALL cases now have identifiable specific
genetic abnormalities with prognostic and therapeutic relevance.121
Although risk classification based on the primary genetic
abnormalities of leukemic cells has great intuitive appeal, its
predictive value is not especially high,123 probably because of
secondary cooperative mutations, host pharmacogenetics and
the over-riding impact of treatment on the prognostic information conveyed by these genetic abnormalities. For example, in
an international collaborative study, Maurizio Aricò and
associates showed that among Ph-positive ALL, an age of 1–9
years and a low presenting leukocyte count confer a relatively
favorable prognosis.124 Ph-positive cases with a leukocyte count
above 100 109/l have an extremely dismal outcome. More
recently, the same group of investigators demonstrated the
clinical heterogeneity among cases with 11q23 abnormalities
(Table 1).125,126 Age was found to be the most important
prognostic factor; infants younger than 1 year fared significantly
worse than patients 1 year of age or older. Among patients with
t(11;19) ALL and MLL-ENL fusion, those with a T-lineage
immunophenotype, who were all over 1 year of age, had a
superior outcome as compared to patients over 1 year of age
with B-lineage ALL.126 Notably, there was heterogeneity even
among infants with t(4;11) ALL; those with a poor early response
to prednisone had an especially dismal prognosis.
Gene expression profiling using DNA microarrays can not
only accurately identify the major, important subtypes of ALL,
but also provide insights into their underlying biology and the
nature of cellular responses to antileukemic therapy.122,127–129
Dr James Downing and colleagues (St Jude Children’s Research
Hospital, Memphis, TN, USA) studied 360 pediatric ALL
patients. Distinct expression profiles identified six important
leukemic subtypes, including T-cell ALL, E2A-PBX1, BCR-ABL,
TEL-AML1, MLL rearrangement, and hyperdiploid 450 chromosomes.127 In addition, another ALL subgroup was identified
based on its unique expression profile. Examination of the
genes comprising the expression signatures provided important
Table 1
The 5-year event-free survival estimates in ALL with
11q23 rearrangements, according to age and phenotype
Type
Category
t(4;11)
o1 year
41 year
B-lineage, o1 year
B-lineage, 41 year
T-lineage
o1 year
41 year
o1 year
41 year
o1 year
41 year
t(11;19)
t(9;11)
del(11)(q23)
Other 11q23
% EFS (s.e.)
19
42
27
46
88
38
46
40
73
22
65
(3)
(5)
(9)
(14)
(13)
(15)
(14)
(22)
(5)
(8)
(7)
P-value
o0.001
0.01
0.27
0.05
o0.001
2371
insights into the biology of these leukemia subgroups. For
example, E2A-PBX1 leukemias were characterized by high
expression of the c-MER receptor tyrosine kinase, a known
transforming gene, suggesting that c-MER may be involved in
the abnormal growth of these cells. Overexpression of FLT-3
receptor tyrosine kinase was detected in MLL-rearranged
leukemias, a finding confirmatory of that recently reported by
Stan Korsmeyer and colleagues.128 On the basis of this finding, a
Phase I pediatric trial of inhibitor of FLT-3 tyrosine kinase will
soon be initiated.
To elucidate the genomics of cellular responses to cancer
treatment, Dr William Evans and colleagues (St Jude Children’s
Research Hospital, Memphis, TN, USA) analyzed the expression
of over 9600 human genes in ALL cells before and after in vivo
treatment with methotrexate and mercaptopurine given alone or
in combination.130 Changes in expression of 124 genes
accurately discriminated among the four treatments. Discriminating genes included those involved in apoptosis, mismatch
repair, cell cycle control and stress response. Only 14% of genes
that changed when these medications were given as single
agents also changed when they were given together. Several
gene clusters responded in a treatment-specific manner. For
example, expression of the ataxia telangiectasia mutated (ATM)
gene was higher after treatment with methotrexate alone but not
after the other treatments. Consistent with G1 arrest by ATM, the
percentage of ALL cells in S phase was lower after treatment
with methotrexate alone but higher after the other three
treatments. Ongoing studies by Dr Mary Relling and colleagues
(St Jude Children’s Research Hospital, Memphis, TN, USA) are
testing the hypothesis that gene expression profile can enhance
our ability to identify patients at risk of developing specific
complications. Preliminary data suggested that supervised
hierarchical clustering and principal component analysis can
identify selected genes expressed by ALL blasts that were
associated with the development of secondary AML or brain
tumors. However, these findings need to be confirmed by
studying independent test sets.
Inherited differences in the metabolism and disposition of
drugs and polymorphisms in the genes that encode drugmetabolizing enzymes, transporters or targets can profoundly
influence the efficacy and toxicity of drug therapy, thereby
affecting the antileukemic response.131 Essentially all genes
encoding drug-metabolizing enzymes responsible for key
modifications of functional groups (phase I reactions) or for
conjugation with endogenous substituents (phase II reactions)
exhibit polymorphism. In general, phase I enzymes activate
endogenous or exogenous substrates, and phase II enzymes
deactivate or detoxify them. Thiopurine methyltransferase
(TPMT) is a phase II enzyme that converts thiopurine prodrugs
into methylated metabolites which are mostly inactive. This
enzyme competes with other enzymes involved in the activation
of thiopurine to their thioguanine nucleotides, which are
incorporated into DNA and induce an antileukemic effect.
TPMT activity is inherited as an autosomal codominant trait.
Approximately 90% of the population are homozygous for the
wild-type allele and have full enzyme activity; about 10% are
heterozygous for the polymorphism and have intermediate
levels of enzyme activity; and one of 300 persons carries two
mutant TPMT alleles and does not express functional TPMT.
Patients with TPMT deficiency accumulate higher levels of
thioguanine nucleotides, and experience more hematopoietic
toxicity when treated with standard doses of mercaptopurine, as
compared to those with normal enzyme activity.132 Not
surprisingly, patients who have deficient TPMT activity also
tend to have better leukemic control than do those with normal
Leukemia
Novel approaches to treat leukemia
RA Larson et al
2372
Leukemia
activity.133 It should be noted that patients who have deficient
TPMT activity are at an increased risk of epipodophyllotoxinrelated AML or irradiation-induced brain tumor, in the context
of antimetabolite therapy.134,135 In fact, therapy-related AML
has also been reported in these patients even when their
treatment consisted primarily of antimetabolites.136
Thymidylate synthase, an essential enzyme in proliferating
cells, is an important target of methotrexate. A homozygous
triple-tandem-repeat polymorphism of the thymidylate synthase
enhancer has been associated with increased enzyme expression and an inferior treatment outcome in childhood ALL.137
The homozygous polymorphism variant (substitution of T for C
at position 677) of methylenetetrahydrofolate reductase, on the
other hand, correlates with an increased risk of oral, GI or
hepatic toxicity following low-dose methotrexate,138,139 and
with greater in vitro sensitivity of leukemic blasts to methotrexate.140
Early response to therapy is one of the most important
prognostic factors because it reflects both the leukemic-cell- and
host-related characteristics. Measurement of MRD, by flow
cytometric detection of aberrant immunophenotype or by PCR
of clonal antigen-receptor gene rearrangements, affords a level
of sensitivity and specificity not attainable by traditional
morphologic assessment of treatment response.141,142 Dario
Campana and colleagues (St Jude Children’s Research Hospital,
Memphis, TN, USA) have shown that patients who attain a
molecular or immunologic remission (defined as leukemic
involvement of less than 0.01% of nucleated bone marrow
cells) after 4–6 weeks of remission induction have a significantly
better treatment outcome than do those who do not achieve this
status.142,143 In fact, almost half of all patients have an MRD
level p0.01% after only 2 weeks of remission induction and
these patients have an exceptionally good prognosis, with less
than 5% cumulative risk of relapse.144,145 By contrast, patients
with an MRD level of X1% at the end of remission induction, a
level that is difficult to detect by morphologic examination,
fared as poorly as those who were considered induction failures
by conventional criteria (ie, X5% marrow blast cells). Tandem
application of flow cytometry and PCR testing allows us to
successfully detect MRD in virtually 100% of cases of childhood
ALL.146 We have therefore incorporated MRD detection into our
current risk assessment system.120 Comparison of paired bone
marrow and blood samples obtained concomitantly disclosed
virtually identical levels from both sources in T-cell ALL but
generally higher levels from bone marrow in B-lineage
ALL.147,148 This finding suggested that T-cell ALL originates
from extramedullary sites (eg, thymus) and subsequently invades
bone marrow, and that T-cell cases can be monitored by
examination of peripheral blood.
While many advances have been made in the treatment of
ALL, only CNS-directed therapy and hematopoietic stem cell
transplantation will be briefly addressed here. Most contemporary clinical trials limit the use and dose of cranial irradiation
because of concern over late sequelae. We recently reviewed
the survival experience of our 10-year event-free survivors and
observed an over 20% cumulative risk of secondary neoplasms
at 30 years from diagnosis among those who had received CNS
irradiation, resulting in a slight excess in mortality rate as
compared to the general population.149 The irradiated patients
also had a lower employment rate and among women, a lower
marital rate. This finding should provide additional impetus to
omit the use of cranial irradiation.
Two studies have omitted cranial irradiation in all patients
regardless of their risk classification, resulting in rates of isolated
CNS relapse of 4.2 and 3% and of any CNS relapse of 8.3 and
Table 2
ALL
Risk factors for traumatic lumbar puncture at diagnosis of
Risk factor
Black
Age o1 year
Early eraa
Less experience
Platelet count o100 109/l
a
Odds ratio (95% CI)
1.5
2.3
1.4
1.4
1.5
(1.2–1.8)
(1.7–3.0)
(1.2–1.7)
(1.1–1.8)
(1.2–1.8)
Lack of dedicated procedure area and general anesthesia.
6%, respectively.150,151 We believe that with more precise
assessment of initial risk factors, and use of optimal intrathecal
and systemic chemotherapy, CNS relapse hazard can be
substantially reduced.152 In this regard, besides high-risk genetic
features and large leukemic cell burden, the presence of
leukemic cells in cerebrospinal fluid (even from iatrogenic
introduction via traumatic lumbar puncture) is also associated
with an increased risk of CNS relapse.153–155 We recently
identified several factors associated with the risk of traumatic
lumbar puncture, including experience of the clinician, platelet
count and the use of sedation or anesthesia (Table 2).156 In our
center, the lumbar puncture is now performed by the most
experienced clinician and under deep sedation or general
anesthesia. We transfuse all thrombocytopenic patients with
platelets before lumbar puncture at diagnosis, and administer
intrathecal chemotherapy immediately after collection of
cerebrospinal fluid.
Autologous transplantation has little if any therapeutic role in
childhood ALL, and the indications for allogeneic stem cell
transplantation from an HLA-matched related or unrelated
donor during first remission of ALL are generally based on
anecdotal evidence. A recent international collaborative study
by Aricò and colleagues124 clearly demonstrated that allogeneic
stem cell transplantation from an HLA-matched related donor is
superior to intensive chemotherapy alone in prolonging initial
complete remission in children with Ph-positive ALL, regardless
of other risk features. However, transplantation did not improve
outcome in patients with t(4;11).125,126 Studies are needed to
determine definitively if transplantation benefits patients with
severe hypodiploidy, near-diploidy or leukemia that has
responded very poorly to initial remission-induction therapy.
Several new agents are being tested. Arabinosylguanine is
quite effective for T-cell ALL but it has narrow therapeutic index
and is associated with neurotoxicity (eg, seizure and somnolence).157 Imatinib mesylate, which inhibits the BCR-ABL fusion
protein and other tyrosine kinases, has induced a response rate
of 70% with 20% complete (albeit transient) in patients with
relapsed Ph-positive ALL,158 and will be tested in newly
diagnosed cases. Other promising drugs include clofarabine159
and FLT-3 inhibitors.129 Further refinements in molecular
classification, together with the identification of leukemic- and
host-related genetic features that affect the efficacy and toxicity
of antileukemic therapy, will afford unique opportunities to
devise treatment plans for individual patients, and thus to further
advance the cure rate.
Questions from audience
Q: I am curious about the data on the MRD. Do you think that
will lead eventually to a change in how these patients are
treated? There is the maintenance phase, which has the potential
for toxicities. It is a heritage of the past where patients were
Novel approaches to treat leukemia
RA Larson et al
2373
treated for a very long period of time. Now we have some
mechanisms to see whether or not their disease is there or not
and do you think that this will be changed in the future.
Ching-Hon Pui, MD: While MRD determination can identify
patients at increased risk of relapse who may benefit from
intensified therapy, I do not think it can be used to determine
when we can stop therapy. ALL uniquely requires long-term
continuation treatment. Although half of the patients have no
measurable disease even by flow cytometry or PCR at the end of
remission induction, all of them will relapse if continuation
treatment is not given.
The Tokyo Children’s Cancer Study Group had carried out an
interesting study that featured only 1 year of therapy, and
yielded an approximately 60% rate of long-term event-free
survival. This finding indicates that 60% of patients may have
therapy stopped at 1 year. However, this group of patients
cannot be reliably distinguished from those who require
prolonged therapy. Unfortunately, MRD determination cannot
be used to guide cessation of therapy. For example, virtually all
patients have negative MRD at the end of 212 years of
continuation therapy but 10–20% of them will relapse. Therefore, MRD cannot be used to determine the length of
continuation therapy. However, it may prove to be useful to
determine the intensity of therapy.
high lysozyme levels and dehydration. Dehydration is a frequent
event in leukemia at diagnosis. All these abnormalities could be
life threatening and obviously TLS has to be prevented or
treated. A diagram of TLS and kidney dysfunction is presented in
Figure 9.
Different types of TLS are observed, for example, spontaneous
TLS can occur before any chemotherapy in rapidly growing
tumors and the best example is Burkitt’s lymphoma. Induced
TLS is also observed in tumors highly sensitive to chemotherapy,
like Burkitt’s, but also in acute leukemia. Tumor burden in acute
leukemia can represent approximately 1 kg of tumor. After 1
week, 99% of the tumor is destroyed, which can create
physiological disorders.
Acute renal insufficiency is due to the renal tubular damage.
Normally, phosphorus, uric acid and calcium are filtered by the
glomerulus, and are partly reabsorbed in the proximal tubules.
In the distal tubules the urine concentrates, and in the collecting
tubules precipitation of uric acid could occur in case of
hyperuricemia, especially due to the acidic pH of the urine.
Also, precipitation of calcium phosphate can occur if the pH is
basic. The situation is worsened when lysozymes also precipitate in tubules. This can occur in a patient with either a
monocytic component or an M4 or M5 leukemia (according to
FAB classification). This precipitation leads to acute tubulopathy, leading to anuria and acute renal failure.
TLS in leukemias: frequency, diagnosis and management
(Jean-Pierre Marie, MD)
Frequency of TLS in adult acute leukemia
TLS is a group of metabolic abnormalities associated with a
rapid cell death in patients with cancer.160–187 All the contents
of the cell will be released in the circulation when the cell dies.
This means that released potassium could lead to hyperkalemia.
Phosphorus release could lead to hyperphosphatemia and
hypocalcemia due to phosphorus–calcium complexes, which
are responsible for nephrocalcinosis. Hyperkalemia and hypocalcemia could be responsible for cardiac arrhythmia.
The DNA present in the cell’s nucleus is degraded in the liver,
which can give rise to hyperuricemia. Hyperuricemia and
nephrocalcinosis are the major disorders associated with TLS;
they could lead to acute renal insufficiency. This renal
insufficiency could be worsened by other factors, including
In the scientific literature it is very difficult to find an article on
the frequency of TLS in adult acute leukemia. Dr Marie
presented a retrospective analysis looking at TLS and its
prevention or treatment in adults treated for acute leukemia
during the last 3 years at the Hôtel Dieu Hospital in Paris.
The files of 73 patients hospitalized at Hôtel Dieu hospital
were reviewed. The mean age was 56. There were 63 AML and
10 ALL patients and the mean WBC count was 35 500. This is
quite representative of the acute leukemia patients hospitalized
at the Hôtel Dieu Hospital. The files of these patients were
reviewed for TLS parameters. The levels of potassium, phosphorus, uric acid and creatinine were examined at the time of
diagnosis before any chemotherapy to ascertain the possibility
Figure 9
TLS and kidney dysfunction. This diagram provides an overview of the effects of TLS on kidney function and the effects of no
treatment vs rasburicase treatment.
Leukemia
Novel approaches to treat leukemia
RA Larson et al
2374
of spontaneous TLS. The patient files were reviewed also during
the first week of intensive chemotherapy, to determine the
frequency of chemotherapy-induced TLS.
TLS at diagnosis
Elevated life-threatening potassium levels were never observed.
Significantly elevated phosphorus levels were observed in 15%
of the cases. With regard to uric acid levels, 18% of the patients
had a significant hyperuricemia. The mean values of creatinine
for women and men according to EDGE were borderline.
However, 26% of the patients presented with elevated
creatinine levels at diagnosis. This could have resulted from
dehydration.
The study at Hôtel Dieu Hospital was unable to find any
correlation between the WBC count and the potassium,
phosphorus, creatinine or uric acid levels. But interestingly,
there was a very good correlation between the creatinine and
uric acid levels.
A correlation between FAB subtype and creatinine levels was
observed. Patients with a monocytic, monoblastic component
(M4 and M5 of the FAB classification) had a higher creatinine
level at diagnosis than the other myeloid leukemia patients.
Lymphoblastic leukemia patients also had higher creatinine
levels than the myeloid non-M4-M5 leukemia patients.
The frequency of chemotherapy-induced TLS was examined.
The variation of this parameter between day 0, before
chemotherapy and during the first week of chemotherapy was
examined. The percentage of variation of these parameters was
examined. A mean variation of þ 11% was observed with
potassium levels, but only four patients with more than a 50%
increase in potassium levels were observed during the first
week. A mean variation of þ 27% was observed with
phosphorus, and 13% of patients had an increase in phosphorus
levels of more than þ 50%. Phosphorus levels probably
represent the best surrogate marker for TLS, because preventative treatments for decreasing phosphorus levels were not
administered. On the other hand, a sharp decrease of uric acid
levels with more than a 50% decrease was observed in the
majority of the patients, and only 5% of patients experienced a
450% increase of serum uric acid level.
With regard to the creatinine levels, the mean variation was
not significantly different before and during chemotherapy, and
two different patient populations were observed. A total of 42%
of patients had a sharp (450%) decrease in creatinine levels.
On the other end, 11% of the patients had a significant increase
(450%) of their creatinine levels. Patients with uric acid and
creatinine increases are the patients developing TLS most
frequently.
In summary, TLS in acute leukemia, according to this
retrospective analysis, is present in about 20% of the patients
at the time of diagnosis. A renal impairment was present in 26%
of the patients, and uric acid and creatinine levels before the
beginning of chemotherapy were highly correlated with TLS.
Treatment of TLS
Measures taken to contain TLS helped avoid major metabolic
disorders during chemotherapy treatment. These measures
consisted of vigorous hydration and alkalinization, control of
the uric acid production as well as its degradation.
Uric acid is a terminal product in human and primate
catabolism of urine (Figure 10). Uric acid is a product of
Leukemia
oxidation of hypoxanthine and xanthine oxidase. Uric acid is
not very soluble, and can crystallize in the urine. In other
species, the terminal product is not uric acid but allantoin
(Figure 10). Allantoin is the degradation product of uric acid by
urate oxidase. Allantoin is five times more soluble than uric
acid, avoiding a precipitation in the tubule. In humans, the urate
oxidase protein is not functional because of a nonsense
mutation.
Prevention of uric acid overproduction relies on
allopurinol
Allopurinol is a very potent inhibitor of xanthine oxidase and it
blocks the formation of uric acid156 (Figure 11). But this drug is
unable to degrade the uric acid already formed. So it would be
very useful to have a recombinant urate oxidase able to destroy
uric acid and to transform it into allantoin avoiding renal uric
acid crystallization.
A nonrecombinant urate oxidase, Uricozymes, was
extracted from Aspergillus flavus and has been available for
over 30 years in France and Italy.188–194 It is widely used to
avoid TLS in patients with Burkett’s lymphoma, ALL and
AML. Severe allergic reactions and anaphylactic shock are
rarely observed. But the limited production of Uricozyme and
its subsequent purification for use in humans precluded its
worldwide distribution.
Recently, rasburicase, a recombinant urate oxidase, has
become available in the USA and in Europe.160,189,195–201 In
the USA, its use is restricted to pediatric patients with tumors.
Compared to the nonrecombinant Uricozyme, this recombinant
product has a greater purity and also a greater enzyme activity.
The increased production of rasburicase has permitted worldwide distribution of its product.
Rasburicase is much more active than allopurinol: 4 h after
the first injection, there was a dramatic decrease of the serum
uric acid levels after rasburicase compared to slow decrease
after allopurinol. At 48 h, there was no difference in serum uric
acid level between allopurinol and rasburicase.
Concerning safety, in a European study of 107 patients, only
3% of adverse events related to the drug were observed. Two
patients had rash and one patient had grade 3 hemolysis, but this
patient was a G6PD-deficient patient.
It was also observed that 7% of the patients generated
antibodies (positive ELISA test), after the drug was administered.
It is important to remember that urate oxidase is a foreign
protein, not synthesized by humans. Three deaths were
observed during the study, but none of them were related to
rasburicase.
The prevention of TLS in leukemic patients involves
heavy hydration and antiuric acid treatment. A total of 77% of
the patients in our series received three or more liters of
hydration plus bicarbonate. Allopurinol was administered in
91% of the cases, often alone but sometimes with urate
oxidase. In all, 29% of the patients received urate oxidase,
sometimes alone but usually in combination with allopurinol.
These measures to prevent hyperuricemia have to be initiated
before chemotherapy in order to prevent increases in levels
of creatinine during chemotherapy. When hyperuricemia
was prevented before chemotherapy, only 10.5% of the
patients showed elevated levels of creatinine during therapy.
On the other hand, close to half of the patients displayed
elevated levels of creatinine when the initiation of prevention
and treatment of hyperuricemia were performed after the
beginning of chemotherapy.
Novel approaches to treat leukemia
RA Larson et al
2375
Figure 10
Purine catabolism and prevention of TLS. This diagram depicts biochemical pathways responsible for degradation of purines
following rapid cell lysis induced by chemotherapy and also illustrates the ability of either purified or recombinant urate oxidase to convert uric
acid to allantoin.
Figure 11
Allopurinol is an inhibitor of xanthine oxidase. This
diagram depicts inhibition of xanthine oxidase by allopurinol to
prevent conversion of hypoxanthine to xanthine to uric acid.
Use of urate oxidase
Urate oxidase was administered to two-thirds of the patients
with increased creatinine levels at initial visit and to one-half of
the patients with a high risk of TLS, such as FAB M4–M5 patients
with lymphoblastic leukemia. The best time to begin urate
oxidase treatment is before day 0 of chemotherapy, usually day
2 or day 3. Sometimes urate oxidase was administered later,
but only as a salvage therapy when unexpected TLS developed.
The levels of uric acid were examined 3 days after initiation of
chemotherapy. A 90% decrease in uric acid levels was observed
in patients treated with urate oxidase (7allopurinol), compared
with a 36% decrease in patients treated with allopurinol alone.
In summary, an early prevention of TLS before day 1 of
chemotherapy reduces the risk of creatinine increases during
chemotherapy. Allopurinol was administered to most patients,
and urate oxidase was given preventively in an emergency
setting to patients with monocytic components, in ALL patients
with a large tumor burden, in patients with increased creatinine
at presentation, and also in patients with high hyperleukocytosis
needing an immediate chemotherapy within 1–2 days.
According to those results, guidelines were proposed for the
prevention and treatment of TLS in adult acute leukemia. In
patients with a low risk of TLS, it is important to start hydration
(1.5 l/m2 including bicarbonates) and allopurinol administration
(600 mg/day) as soon as possible, usually 2 days before
chemotherapy and until WBC decrease to normal. This
Leukemia
Novel approaches to treat leukemia
RA Larson et al
2376
treatment is sufficient to control hyperuricemia and to prevent
increase of creatinine levels in 75% of the cases.
For patients who develop hyperuricemia despite this treatment, it is important to increase hydration and administer urate
oxidase at 0.2 mg/kg/day until uric acid is definitively controlled. Usually, 2–3 days are enough to reduce uric acid levels
to normal.
In patients with high risk of TLS or even if TLS is already
present or highly anticipated, it is important to begin as soon as
possible before chemotherapy with vigorous alkaline hydration,
2–3 l/m2/day. Urate oxidase (0.2 mg/kg/day) is administered
during the first few days of chemotherapy.
In conclusion, TLS was found in roughly 20% of adult patients
with acute leukemia. The main risk is acute tubulopathy, caused
mainly by crystallization of uric acid. Urate oxidase catalyzes
the conversion of uric acid to allantoin, avoiding tubular
crystallization. TLS can be prevented through hydration plus
antiuric acid treatment, mainly allopurinol or in selected
patients, urate oxidase. Severe TLS has to be treated/prevented
with vigorous hydration, and urate oxidase.
Summary
In this meeting report, exciting new therapies for leukemia
treatment were discussed. Through molecular biology and
cytogenetic approaches, many of the genes responsible for
transformation of hematopoietic cells have been identified. Due
to the elucidation that aberrant expression of certain oncoproteins causes cancer, fundamental increases in our understanding
of the mechanisms of leukemogenesis have been obtained,
which has yielded many novel therapeutic approaches. Many of
these oncogenes activated in human cancer are created by the
fusion of genes by chromosomal translocation. These translocations result in two broad classes of oncoproteins, kinases (eg,
BCR-ABL, TEL-JAK) or transcription factors (TEL-AML-1, PMLRARa) with modified activities. Some oncogenes are activated
by point mutations (RAS) or small amplifications of certain
regions of the gene (FLT-3). Some chromosomal translocations
(BCR-ABL, TEL/PDGFbR) may occur singly in myeloproliferative
disorders; however, complimenting translocations must occur
for the transition to acute leukemia (NUP98/HoxA9, AML1/
EVI1, AML1/ETO).
More hope has been brought into the treatment of leukemia
by the characterization of small molecular weight cell membrane permeable signal transduction pathway inhibitors. Gleevec is an inhibitor of BCR-ABL that can have astounding results
on the chronic phase of CML with 495% normalization of
blood counts. Other inhibitors have been developed that target
the FLT-3 and Ras pathway (FLT-3, Raf and MEK inhibitors).
These inhibitors may be especially useful in the treatment of
CML patients whose leukemia cells have become resistant to
Gleevec. Another class of inhibitors is represented by RA, which
acts as a differentiating agent and can be effective in treating
APL patients with PML-RAR chromosomal translocation.
Furthermore, as more is known about how normal and chimeric
transcription factors work, it has been clear that compounds that
inhibit histone deacetylation (HDAC inhibitors) may prove
effective in certain leukemia therapies. These compounds may
prevent the block in differentiation induced by the chimeric
transcription factor oncoproteins.
Drug resistance remains a nagging problem in cancer
chemotherapy. Eukaryotes have evolved pumps to remove
toxins they encounter in their environments. Hematopoietic
precursor cells may be particularly resistant to many chemotherLeukemia
apeutic drugs, as they are ‘designed’ to survive repeated
exposure to heterogeneous toxins. The overexpression of one
of these pumps, P-gp, is detected in AML blasts, particularly in
older patients, and may be increased at relapse. P-gp overexpression is associated with a poorer outcome. Modulators of
the drug pumps have been discovered. These modulators (eg,
cyclosporine, PSC-833) can reverse the effects of the drugresistant pumps. Thus, it is may be possible to treat patients with
lower concentrations of the chemotherapeutic drug and the
modulators. The goal of these studies with drug pump
modulators is to raise the intracellular chemotherapeutic drug
levels in the leukemic cells to a greater extent than can be
achieved by administration of the chemotherapeutic drug by
itself. A problem remains that these ‘drug pumps’ also serve
important physiological roles in their normal tissues. Thus there
is often toxicity associated with interference with the normal
functions of the drug pumps.
CLL is the most common type of leukemia observed in the
Western Hemisphere with over 10,000 cases/year and is not
curable with currently available therapy. Most patients are
initially asymptomatic but eventually require therapy. Until the
advent of monoclonal antibodies, the therapeutic options
included treatment with chlorambucil, prednisone and purine
analogs. Fludarabine is the best single drug for initial treatment
and results in improved response rates and progression-free
survival. However, there exists significant opportunities to
improve fludarabine therapy. Humanized antibodies have been
generated to treat CLL. The humanized antibodies have led to
improved efficacy and diminished toxicity and represent new
hope in the treatment of CLL. These humanized antibodies are
directed at different proteins including CD22, CD23, CD52,
HLA-DR and they exert their effects by different mechanisms
including ADCC, CDC and apoptosis. Some of these antibodies
will induce caspase 3-dependent (Rituxan) and caspase 3independent (Hu1D10) apoptosis. Hu1D10 induces apoptosis in
CLL cells and it does not induce ADCC or CDC, and the
apoptosis induced by Hu1D10 is caspase independent but leads
to the production of AIF release, which may involve changes in
the mitochondrial membrane potential. The apoptosis induced
by Hu1D10 may occur by inhibition of Bcl-2 function leaving
many of the other apoptotic machinery intact, as overexpression
of Bcl-2 is protective and neutralizes the apoptotic effects of
Hu1D10. Many other monoclonal antibodies have been
developed to treat CLL and are currently being evaluated. Some
of the antibodies target the CLL microenvironment and include
aTNF, aVEGF, aIL4, aSDF and abFGF. Other antibodies are
conjugated to toxins (aCD22–diptheria toxin, aCD19–genestein)
or some are radiolabeled (aHLA-DR-I131, aCDC20Y90, aCD22,
aCD52I131).
There remain many challenges to improve the survival rate in
ALL. The type of chromosomal translocation the patient has and
the type of hematopoietic cell that the lesion is present in often
determines how long a patient will have event-free survival.
Leukemogenomics is now a real concept as gene expression
profiling studies performed at St Jude Children’s Research
Hospital have led to exciting methods that can classify patients
into particular leukemia subtypes and can also predict whether
those patients will have relapses over a given time period. This is
also being coupled with determining the patient’s genotypes for
genes involved in intermediary metabolism, drug metabolism,
drug transport, drug receptor, host susceptibility and disease
pathogenesis. Since many of these are enzymes involved in drug
metabolism, these differences can affect the patient’s outcome
after treatment with a particular drug. Thus leukemogenomics
and pharmacogenetics will eventually provide the physician
Novel approaches to treat leukemia
RA Larson et al
2377
with the proper drug and concentration to treat individual
leukemia patients.
Complications of TLS can be prevented by either allopurinol
or rasburicase treatment. This meeting also discussed the results
of recent clinical trials designed to prevent TLS by either
allopurinol or rasburicase treatment. Clearly, rasburicase is
more effective and rapid in reducing uric acid levels resulting
from TLS than allopurinol. As a result of the cloning of urate
oxidase and the subsequent manufacturing of this recombinant
protein, clinicians will be able to treat potential TLS patients
with rasburicase. Proper identification of patients of high risk for
TLS and their subsequent treatment with rasburicase will reduce
renal problems associated with TLS. Adverse immune reactions
resulting from administration of a foreign protein may develop if
cancer patients are re-treated with rasburicase. However, this
may not be a major problem if patients are cured of their cancer
after a single treatment. Rasburicase is an intriguing example of
how basic science can be applied to prevent a disease. A
recombinant enzyme absent in humans is infused to catabolize
uric acid that could otherwise accumulate to toxic levels in
certain disease states.
Cancer research has surpassed many important milestones in
the past 30 years. These advances have stemmed from
technological discoveries that have allowed the cloning of
DNA sequences, the discovery of oncogenes and their roles in
human cancer, the knowledge of how transcription factors,
promoters and chromatin structure affect gene expression, the
sequencing of the human genome, and the discovery and
clinical applications of monoclonal antibodies. Many scientific
advances have been achieved by brute-force screening of
pharmaceutical libraries. This has led to numerous clinical
trials, which have tested the effects of particular compounds or
antibodies to suppress cell growth, which might under certain
circumstance cure the leukemia patient. Undoubtedly and most
often, these drug screening and clinical trials yielded negative
results. But we must always be optimistic with the advances that
the cancer field has made, and continue to test possible
mechanism and therapies. Unquestionably, advances have been
made that have improved the quality of care and the extended
survival of leukemia patients.
Acknowledgements
GQ Daley was supported in part by the Grants NIH CA76418,
CA86991, DK59279 and HL71265, and a sponsored research
agreement from the Schering-Plough Research Institute. GQ
Daley is a Birnbaum Scholar of the Leukemia and Lymphoma
Society of America. P Porcu was supported in part by a grant
(K23CA102155) from the NCI. C-H Pui was supported in part by
the Grants CA 21765, CA 31566, CA 51001, CA 78824, CA
29139, CA 37379 and GM 61393 from the US National Institutes
of Health; a Center of Excellence grant from the State of
Tennessee, USA; and the American Lebanese Syrian Associated
Charities. C-H Pui is the American Cancer Society FM Kirby
Clinical Research Professor. JA McCubrey was supported in part
by the Grants CA 51025 and CA098195.
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