The Comprehensive Guide to Banff:
Understanding the VIIIth International Myeloma Workshop
Published by:
INTERNATIONAL MYELOMA FOUNDATION
12650 Riverside Drive, Suite 206
North Hollywood, CA 91607-3421 U.S.A.
Tel: 818·487·7455 Fax: 818·487·7454
E-mail: TheIMF@myeloma.org
Website: www.myeloma.org

---

TABLE OF CONTENTS
INTRODUCTION
1
Susie Novis
THE MALIGNANT CLONE IN MYELOMA
2
Herve Avet-Loiseau, M.D.
ONCOGENIC EVENTS
4
Thomas E. Witzig, MD
GENETIC INSTABILITY AND ONCOGENESIS
6
Johannes Drach, M.D.
INNOVATIVE TECHNOLOGIES
8
Brian Van Ness, Ph.D.
SIGNALING AND MICROENVIRONMENT
10
Dharminder Chauhan, Ph.D.
ANGIOGENESIS IN MULTIPLE MYELOMA
12
Ivan Van Riet, Ph.D.
TREATMENT MODALITIES AND SYMPTOM MANAGEMENT
14
Robert A. Kyle, M.D.
BONE METABOLISM, BONE DISEASE, AND THE BISPHOSPHONATES
16
Gregory R. Mundy, M.D.
ALLOGENEIC AND AUTOLOGOUS TRANSPLANTATION
18
Jesus San Miguel, M.D.
THALIDOMIDE: CLINICAL OUTCOMES
20
Brian G. M. Durie, M.D.
VACCINES STRATEGIES IN MULTIPLE MYELOMA
22
Håkan Mellstedt, M.D., Ph.D.
IMMUNOTHERAPIES AND CONTROL OF RESIDUAL DISEASE
24
Brian G.M. Durie, M.D.
NEW THERAPIES
26
Kenneth C. Anderson, M.D.
INTERNATIONAL MYELOMA FOUNDATION
28
Programs and Services

---

INTRODUCTION
Susie Novis
International Myeloma Foundation
We encourage you to share The Comprehensive Guide to
Banff with your treating physician so that you may both
benefit from the many exciting developments that emerged
from the latest International Myeloma Workshop.
In the coming months, many of these topics will be covered
in greater depth in the IMF newsletter, Myeloma Today.
We will explore the implications for future research and
new treatment strategies. We will address topics ranging
from "What causes myeloma" to "New approaches to classi-
fication and prognostic assessment" to results with new
Dear IMF Member,
therapies.
The International Myeloma Foundation (IMF) is proud to
If you have specific questions about the material covered
present The Comprehensive Guide to Banff: Understanding the
in The Comprehensive Guide to Banff, please contact the
VIIIth International Myeloma Workshop.
IMF and answers will be sought and provided. Any generic
follow up issues wll be posted on the IMF website at
The International Myeloma Workshop is a bi-annual
www.myeloma.org.
medical meeting dedicated exclusively to multiple myelo-
ma. The Workshop is an opportunity for leading myeloma
As always, we thank you for your support of the myeloma
experts to come together and exchange information regard-
community and of the International Myeloma Foundation.
ing the latest developments in the treatment, management
and prevention of myeloma.
Sincerely,
Susie Novis
The IMF recognizes that, while the meeting is intended
President
primarily for clinicians and researchers, this information is
of great interest to everyone within the myeloma communi-
ty. As always, we are working hard to ensure that you,
our valued members, are kept abreast of any and all new
information.
The Comprehensive Guide to Banff provides concise sum-
maries of all the major presentations made at the VIIIth
International Myeloma Workshop. For the readers' conven-
ience, the material has been organized to cover the basic
scientific material, followed by first conventional then more
experimental approaches to treatment.
The Comprehensive Guide to Banff has been made possible,
in large part, thanks to the time and talent of the myeloma
experts who each agreed to author a summary paper detail-
ing the workshop session in which they were involved.
While each paper has been edited for clarity and consisten-
cy, we have strived to maintain the individual "voice" and
writing style of each author. As a result, The Comprehensive
Guide to Banff is meant to read as a collection of individual
summary papers.
1

---

THE MALIGNANT CLONE IN MYELOMA
Herve Avet-Loiseau, M.D.
Institut de Biologie, Nantes, France
the pro-B cell rearranges first the D (diversity) and J (junc-
tion) segments, and then the V (variable) with DJ regions.
Analysis of selected VH segments in more than 100 patients
revealed a bias in the selection. Whereas the V4-34 gene is
selected in 5% to 10% of normal B cells, it has never been
observed in myeloma patients. Analysis of the somatic muta-
tion pattern revealed that myeloma cells are invariably
mutated, without intraclonal variation, with a pattern pro-
viding evidence for antigen selection. However, molecular
analyses of tumor cells from hairy-cell leukemia (an ontoge-
netically closely related malignancy) have shown that these
cells are able to form different clonally related transcripts,
including IgM transcripts. This situation is currently
HIGHLIGHTS
observed in myeloma, but it may represent another proof
against the implication of clonotypic B cell precursors in
The first speaker, Dr. Linda Pilarski of the University of
myeloma pathogenesis.
Alberta, presented data generated in her laboratory on the
origin of the myeloma cell. Myeloma cells are typically plas-
Dr. Marleen Bakkus (Free University Brussels, Belgium)
ma cells, ie, the most differentiated B cells. Her hypothesis is
addressed the same topic (ie, molecular IgH rearrangement)
that these malignant plasma cells derive from clonotypic B
but in a mouse model of multiple myeloma (MM). A specific
cells, which would represent the myeloma progenitors. The
mouse strain (C57BL/KaLwRij) spontaneously develops, in a
major questions regarding these B cells are: (1) do they really
small percentage of cases, a disease that closely resembles
persist in myeloma patients? and (2) if so, what is their con-
humanMM. Several clones (the 5T clones) have been gener-
tribution to myeloma disease? These clonotypic B cells are
ated and then propagated in this mouse strain. Two specific
characterized by a molecular rearrangement identical to that
clones, the 5T2 and the 5T33, reflect different forms of the
of the malignant plasma cells, but they express immunoglob-
human disease, offering an experimental system for studying
ulin (Ig)M rather than IgG or IgA. Dr. Pilarski reported on a
the molecular IgH rearrangements. These two clones express
clinical study with 29 patients that showed a significant cor-
monoclonal IgG. Using sensitive PCR assays, researchers
relation between the presence of clonotypic B cells and
have isolated isotype switch variants (ie, IgA or IgM) in the
reduced patient survival. A correlation between the number
bone marrow of mice grafted with the two clones. Of note,
of plasma cells within the bone marrow at diagnosis and a
the analysis of 5T33 cells growing in vitro failed to detect
higher level of serum b2-microglobulin was observed in this
such isotype switch variants, indicating that in vivo phenom-
small series. Moreover, these cells are detected all along the
ena, such as interactions with the bone marrow microenvi-
disease course, even in patients receiving high-dose thera-
ronment, are crucial for the appearance of these variant tran-
pies. Several hypotheses can explain the persistance of IgM
scripts. This model clearly suggests that these isotype variants
clonotypic cells: (1) a single B or plasma cell may express
originate from malignant myeloma cells (and not the oppo-
both pre- and postswitch isotypes ; (2) small populations of
site) through complex molecular events (such as trans-
pre- and post-switch memory myeloma B cells may circulate
switching or downstream-switching) and does not support
in the blood ; and (3) the myeloma progenitor may be a
the existence of pre-switched myeloma precursors.
postgerminal center IgM-expressing memory B cell that
undergoes persistent, directed isotype switching and terminal
The next speaker was Dr. Leif Bergsagel of Cornell Medical
differentiation to myeloma plasma cells. In other words,
Center in New York. Based on analysis of the molecular IgH
these chemoresistant clonotypic B cells would represent a
breakpoints observed in 40 myeloma cell lines, Dr. Bergsagel
reservoire for the malignant clone and should be targeted by
has developed a model for the oncogenesis ofMM. His group
therapy to stop this process. It was suggested that these IgM
previously showed that most (if not all) human myeloma cell
transcripts could be produced by the malignant plasma cells
lines display illegitimate IgH rearrangements. Most of these
themselves, since most malignant plasma cells retain one Cµ
breakpoints involve switch regions (located on the 5' side of
locus. These provocative results are still in debate.
each constant domain except Cd), whereas in some cases,
the breakpoints occur in the JH region. These results are
The next speaker, Dr. Surinder Sahota, (University of
consistent with the hypothesis that these translocations have
Southamptom, U.K.) focused on molecular rearrangement of
been mediated by errors during the switch recombination
the Ig genes, esssentially IgH (encoding the Ig heavy-chain
and somatic mutation processes and may represent primary
gene). During plasma cell ontogeny, this gene is rearranged
events in the transformation process. However, some myelo-
through several molecular events. Within the bone marrow,
ma cell lines present IgH rearrangements occurring in other
2

---

sites. A detailed analysis of the 40 cell lines reveals striking
features. One observation was that about 40% of the tumors
retain the Cµ gene. This finding supports the hypothesis of
possible trans-switching events, able to generate IgM tran-
scripts, and constitutes experimental proof against the
hypothesis of clonotypic IgM B-cell myeloma precursors.
Analysis of the translocation partners in myeloma cell lines
revealed three main regions: 4p16 (FGFR3/MMSET), 11q13
(CCND1), and 16q23 (c-maf). Cloning of the breakpoints in
these cell lines has shown that they uniformly fall within JH
or switch regions. In contrast, cloning of translocations with
8q24 (c-myc) or other sporadic chromosomal regions has
shown that they almost systematically fall outside the JH and
switch regions. The latter translocations would not be medi-
ated by B-cell-specific recombination mechanisms but may
result from other processes such as genomic instability. Dr.
Bergsagel suggested that the former translocations would be
primary oncogenetic events, whereas the second ones would
represent random secondary events. His group also analyzed
the cell lines for other recurrent genetic events such as muta-
tions of N- and K-ras, observed in 40% of cases ; however,
these events were never observed in cell lines harboring a
mutated form of the translocation t(4;14).The group pro-
posed an oncogenetic model based on analysis of the illegiti-
mate IgH recombination breakpoints, separating primary and
secondary events. This model needs to be demonstrated in
patients' primary tumors.
The final speaker, Dr. Malcom S. Moore (Memorial Sloan-
Kettering Cancer Center) discussed telomerase activity in
myeloma cells. Telomerase expression is normally restricted
to a few cell types, such as fetal or germinal cells. In somatic
cells, telomerase activity is rapidly repressed after birth.
Without telomerase activity, somatic cells exhibit progressive
loss of telomeric sequences with each cell division and finally
enter senescence when a critical length of telomeres is
reached. In contrast, malignant cells are capable of escaping
the senescent checkpoint and undergoing unlimited prolifer-
ation. Indeed, activation of telomerase is widely observed in
human malignancies. Dr. Moore reported on telomerase
activity in highly purified myeloma cells. A wide range of
telomerase activity was observed among 135 samples, reflect-
ing the heterogeneity of myeloma. In contrast, in a few indi-
viduals with monoclonal gammopathy of undetermined sig-
nificance (MGUS), telomerase activity was constantly low.
Telomerase activity was linearly correlated with a number of
disease variables, especially the number of plasma cells and
poor-prognosis cytogenetic abnormalities. This parameter
could represent a novel prognostic tool for patients with MM.
This session was extremely rich and highlighted the com-
plexity of the oncogenesis ofMM. Rearrangements of the IgH
locus appear to be very powerful for the understanding of
these processes. However, most of the results obtained so far
have been generated in mouse or in myeloma cell lines,
which may not perfectly reflect the situation in patients. The
next step will be to focus on patient tumors to gain further
insight into the oncogenesis of MM. v
3

---

ONCOGENIC EVENTS
Thomas E. Witzig, MD
Mayo Clinic, Rochester, Minnesota
is located. When a B cell is developing, it first makes IgM;
later, when it matures into a PC, it switches its production to
IgA or IgG. The region of 14q32 where these translocations
occur is termed the "switch region."
Early in the development of a B cell, sequential, regulated
DNA rearrangements are mediated by joining of segments of
the VDJ genes. This process, referred to as "VDJ recombina-
tion," serves to generate IgH and IgL receptors. Later in
development an antigen stimulates B cells to undergo IgH
switch recombination, primarily in GC B cells, and also
somatic hypermutation, which occurs exclusively in GC B
cells. Mistakes in any of these three B-cell­specific DNA
INTRODUCTION
modifications can result in parts of chromosomes being
moved to other chromosomes. In addition, somatic hyper-
The cause of multiple myeloma (MM) remains unexplained.
mutation can produce minor changes in small portions of the
However, important advances in the understanding of the
DNA, remove a section of DNA, or insert additional seg-
genetic defects found in myeloma cells have been made.
ments of DNA into a strand. These events can lead to
Syndecan-1 is found on the cell surface of most cells in the
abnormal cell activity and, hence, malignancy.
myeloma clone and binds growth factors such as fibroblast
growth factor (FGF)-2. These findings provide important
Dr. Michael Kuehl's research at the National Cancer
clues as to how the plasma cell becomes malignant and how
Institute has determined that IgH translocations occur often
it causes disease in patients.
in myeloma, so that more than 90% of myeloma cell lines,
60% to 70% of advanced myeloma samples, and approxi-
HIGHLIGHTS
mately half of MGUS tumors have at least one IgH translo-
cation. Translocations involving the light-chain locus are
When B lymphocytes are made by the bone marrow, they are
less frequent, so that about 20% of advanced myeloma
stimulated by antigen in the lymph nodes into two kinds of
tumors and cell lines have IgG lambda translocations, but
plasma cells--the pregerminal center plasma cell (pre-
only a low percent of tumors and cell lines have Ig kappa
GCPC) and the post-germinal center plasma cell (post-
translocations. Studies of translocation breakpoints have
GCPC). Pre-GCPCs are usually short lived. In contrast,
determined that each of five chromosomal loci are involved
post-GCPCs continue to make changes in their genetic
in 5% to 20% of myeloma tumors: 11q13 (cyclin D1),
structure so that they can bind even better to the antigen for
4p16.3 (FGFR3+MM.SET), 16q23 (c-maf), 6p21 (cyclin
which they are specific. These post-GCPCs become the
D3), and 8q24 (c-myc). The translocations involving
long-lived PCs and typically migrate back to the bone mar-
4p16.3, 6p21, 11q13, and 16p23 have 14q32 breakpoints in
row, where they live for months to years and manufacture
or near the IgH switch regions or, less often, JH sequences
their immunoglobulin (Ig). MM and monoclonal gammopa-
consistent with errors in IgH switch recombination and
thy of undetermined significance (MGUS) are tumors of
somatic hypermutation. In contrast, translocation break-
long-lived post-GCPCs.
points involving 8q24 and other loci often have 14q32
breakpoints that are not near either IgH switch regions or JH
It is not clear what causes a long-lived PC to begin to prolif-
sequences. Abnormal regulation of c-myc is important in
erate and turn into active MM. It is unusual for patients'
Burkitt's non-Hodgkin's lymphoma and murine plasmacytomas.
MGUS to turn into MM (approximately 1% per year). One
Please see table on page 7.
factor that is likely to be involved is instability of the genes
in the PCs, referred to as "genetic instability." This can occur
Dr. Ho et al of the Royal Prince Alfred Hospital in Australia
when B cells undergo three B-cell-specific DNA modifica-
reported that they had studied myeloma cells from 21
tion processes that generate double-strand DNA breaks ­ V
patients (4 with stable MM, 17 with relapsing MM) and
(variable) D (diversity) J (joining) recombination, IgH
found translocations into the switch regions that were "ille-
switch recombination, and somatic hypermutation. Errors in
gitimate" (ie, not supposed to be there) in 57% (1 of 4 stable
any of these can produce a translocation.
MM, 11/17 relapsing MM). Multiple switch regions were
often involved in one tumor. These investigators then ana-
Translocations of parts of one chromosome to another chro-
lyzed two of the genes that were most commonly involved in
mosome are common in MM. Usually, one partner of the
the translocations: 11p13 and 4p16. In the translocation
exchange is a region on chromosome 14q32 where the genet-
11;14, the cyclin D1 (CD1) gene could be transferred to
ic code for the heavy-chain portion of the antibody molecule
14q32 and the myeov gene, which remains on 11, can func-
4

---

tion abnormally because of the translocation. In the case of
FGFs can stimulate some cells to grow and multiply, whereas
4p16, the FGFR3 is transferred to 14q32, and MMSE is
in other situations FGFs may cause the cell to die or to stop
abnormally regulated on chromosome 4. They studied eight
growing. There are currently 22 known human FGFs that
patients; 2 of them had CD1 expression, and one had
can interact with one or more of the four known FGF recep-
increased myeov expression.
tors (FGFRs) present on the cell surface. When an FGF
binds to its FGFR, a signal is generated from outside the cell,
When disease course was examined, it was found that the
through the FGFR to the inside of the cell. This sets off a
presence of a switch translocation did not affect how long a
cascade of reactions inside the cell that culminates in
patient lived (prognosis). Thus, the translocations cannot
changes in the cell directed by the cell nucleus.
always be found, and they may not necessarily be the initial
problem in the cell but may affect behavior of the cell later
There are four reasons to study FGFs and FGFRs in MM:
in the course of MM. The analysis of switch translocations is
an important research tool; however, it is not currently help-
1)
Angiogenesis (new blood vessel formation) occurs in
ful in treatment planning for an individual patient.
the marrow of patients with active MM but not in
marrow from patients with MGUS. The knowledge
Chromosomal structure and stability is a critical factor in the
that some FGFs are potent stimulators of endothelial
pathogenesis of MM. The work of Drs Kuehl and Ho, along
cells (the cells that make up new blood vessels)
with others, is providing very useful insights into the role of
provides rationale to learn whether myeloma cells
these segments of chromosomes that are activated by move-
make FGFs.
ment of parts of one chromosome onto parts of others. This
is helpful in understanding the underlying mechanisms of
2)
About 10% to 20% of patients with MM have a
how myeloma is initiated and advances. It is likely that a
t(4;14)(p16;q32) translocation in the MM cells.
better understanding of the genetic machinery of the myelo-
The part of chromosome 4p16 that is moved to
ma cell will eventually lead to better methods of diagnosis
chromosome 14q32 contains the genetic code for
and treatment.
FGFR3. Moving FGFR3 to the region of chromosome
14q32 (the area where the genetic code for antibody
A study of the cell-surface proteins on PCs can also explain
formation is contained) results in overexpression of
the behavior of malignant PCs. Syndecan-1 (also known as
FGFR3 in these cells. This may be one way to turn
CD138) is found on the surface of almost all myeloma cells,
a PC malignant and suggests a way that FGFs binding
whether they are in the bone marrow or the blood.
to these FGFR3 receptors (the antenna-like structures
However, syndecan-1 is more than a marker of PCs; rather, it
that extend from the surface of the cell) may stimulate
is a master regulator of molecular encounters. The myeloma
cell growth.
cell can use syndecan-1 to attach itself to other myeloma
cells or to other cells in the bone marrow. The syndecan-1 is
3)
Syndecan-1 is a cell-surface receptor that can be
connected to the inside of the cell to allow the cell to move
identified by anti-CD138 antibody. Studies have
around the bone marrow. Many myeloma investigators use
shown that most PCs (benign and malignant) have
beads with antibodies to syndecan-1 attached to help isolate
syndecan-1. Since syndecan-1 binds FGF, this
PCs in the laboratory. Dr. Ralph Sanderson's group at the
association provides a clue that FGF may play a role
Arkansas Cancer Research Center has shown that syndecan-
in MM.
1 is a binding partner to SP17, a heparan-binding protein
originally thought to be found only in the testes. SP17 is
4)
Thalidomide is an important new treatment for MM.
found on the surface of myeloma cell lines and patient cells
Understanding how thalidomide works will provide
and can bind to syndecan-1 on the cell surface and promote
insight into other ways to kill myeloma cells. We
myeloma cell-to-cell adhesion. The myeloma cell tends to
know that if a pregnant woman takes thalidomide it
organize the syndecan-1 at the leading edge of the cell
can interfere with limb development in the fetus. It
referred to as the uropod. Other factors important to growth
turns out that FGFs are critical components of the
of PCs are also localized at the uropod. Syndecan-1 can be
cell machinery used to form limbs in a developing
shed from the surface of the cell into the serum by the action
fetus. This is a hint that perhaps thalidomide works
of enzymes called proteases. Studies have shown that synde-
in MM by interfering with FGF/FGFR interaction.
can-1 accumulates in the serum of myeloma patients, and
high levels of syndecan-1 in the serum correlate with a poor
A first step in understanding the role of FGFs and FGFRs in
prognosis. Cells that express the soluble form of syndecan-1
MM is to determine whether myeloma cells make
are twice as invasive in the laboratory as cells that do not
FGFs/FGFRs. Human myeloma cell lines are made from
express any syndecan-1 at all. Syndecan-1 can also accumu-
myeloma cells from patients with the disease. The cells are
late within the scarred area of bone marrow of patients with
placed in culture in the laboratory and can be grown and
myeloma cells. This deposit of syndecan-1 could provide a
used for experiments for many years. They are very useful,
reservoir for growth factors that are bound to syndecan-1,
because the investigator can perform many experiments
such as FGF-2.
without having to go back and ask the patient for another
FGFs are proteins that are capable of multiple functions.
Please see page 7
5

---

GENETIC INSTABILITY AND ONCOGENESIS
Johannes Drach, M.D.
University of Vienna, Vienna, Austria
switch recombinations). Interestingly, translocation partners
are remarkably heterogeneous, with chromosomal regions
11q13, 4p16.3, and 16q23 being commonly involved in these
translocations. These chromosomal sites harbor genes that
may function as oncogenes, growth factors, or transcription
factors and thus upon dysregulation could contribute to
myeloma pathogenesis.
Dr. Herve Avet-Loiseau and coworkers from the Institut de
Biologie presented results on 14q32 translocations by inter-
phase cytogenetic analysis (fluorescence in situ hybridization
[FISH]) of primary tumor cells from 700 patients with MM.
Evidence for a 14q32 translocation was obtained in 73% of
these patients. As far as the type of translocation is con-
INTRODUCTION
cerned, 16% had t(11;14), 10% t(4;14), and 2% t(14;16).
However, in the remaining 45% of patients, other chromoso-
In this session, recent information on the occurrence and sig-
mal regions must be involved in the translocation, again
nificance of critical chromosomal abnormalities in primary
underlining the marked heterogeneity of these aberrations.
myeloma specimens was presented. Data highlighted the
Of note, the presence of t(4;14) or t(14;16) was strongly cor-
marked heterogeneity of translocations involving the
related with the presence of other parameters indicating poor
immunoglobulin heavy-chain (IgH) gene locus on chromo-
prognosis (in particular, del 13q). In contrast, such a correla-
some 14q32 as well as prognostic implications of a chromo-
tion was not observed for t(11;14). The investigators pro-
some 13q deletion (del 13q).
posed that MM could be subclassified based on the type of
14q32 translocations; validation of this model will be per-
HIGHLIGHTS
formed during the analysis of ongoing clinical trials.
Genetic instability and chromosomal aberrations leading to
Results presented by Dr. Rafael Fonseca and coworkers, from
dysregulated expression of critical genes are a hallmark of can-
the Mayo Clinic, are in good agreement with the French
cer cells. In recent years, there has been increasing evidence of
data: In their analysis, t(11;14) was present in 53 of 336
an important role for specific chromosomal abnormalities in
patients (16%), and there was no correlation with del 13q.
the biology of monoclonal gammopathies. Moreover, cytoge-
Patients with t(11;14) were more likely to respond to stan-
netic abnormalities are being recognized as powerful prognos-
dard-dose chemotherapy, and there was a trend for longer
tic indicators in multiple myeloma (MM). The session on
event-free and overall survival of these patients. In contrast,
Genetic Instability and Oncogenesis covered both aspects, and
presence of t(4;14), which was detected in 17 of 165 patients
fairly mature data now exist regarding 14q32 translocations
(10%) with MM, was associated with del 13q (present in 16
and del 13q in primary specimens from patients with MM.
of the 17 patients) and shorter survival (23.9 months for
patients with t(4;14) versus 41.6 months for patients lacking
Translocations involving the IgH focus on chromosome 14q32
t(4;14)). Poor outcome was also observed for 11 patients (out
of 216) who presented with t(14;16) [3].
After contact with an antigen, normal B lymphocytes under-
go a maturational process that results in the generation of a
Del 13q
population of short-lived plasma cells pr oducing IgM. Upon
further stimulation, activated B lymphocytes migrate through
This chromosomal abnormality has attracted much attention
the follicle centre of lymph nodes, where somatic mutations
since the original report by Tricot et al., who demonstrated
of the variable sequences of the IgH gene occur, which even-
short event-free and overall survival for MM patients with
tually leads to a generation of cells with high-affinity anti-
partial or complete del 13q as analyzed by metaphase cytoge-
body production. Finally, plasmablasts undergo IgH switch
netics. By means of interphase FISH, the frequency of del
recombination. During this process, small segments of DNA
13q is even higher than detected by metaphase cytogenetics,
within the IgH gene are juxtaposed, which results in a switch
and the data presented are quite consistent: A del 13q was
of the produced Ig from IgM to another isotype (most com-
present by FISH in 102 of 245 patients (42%) in our series,
monly IgG or IgA). Thus, already under physiologic circum-
in 176 of 325 patients (54%) in the analysis by the Mayo
stances, the IgH locus at chromosomal band 14q32 repre-
Clinic, and in 286 of 669 patients (43%) in the analysis by
sents a chromosomally unstable region.
the French study group. It was also a consistent finding in all
three series that presence of a del 13q as analyzed by FISH
Dr. Leif Bergsagel and colleagues of Cornell Medical Center
confers a poor prognosis. This was true for both standard-
demonstrated that translocations involving the switch region
dose chemotherapy and high-dose melphalan with autolo-
of 14q32 are an almost universal event in myeloma cell lines.
gous stem cell support, and del 13q remained an independent
However, unlike the physiological process, IgH sequences ar e
prognostic parameter on multivariate analysis. In the analysis
rearranged with non-Ig gene sequences (so-called illegitimate
presented by Dr. Avet-Loiseau, del 13q and a serum b2-
microglobulin (b2M) >3 provided powerful prognostic infor-
6

---

mation: Depending on the number of risk factors present (0
Oncogenic Events -
versus 1 versus 2), patients with good, intermediate, and
Continued
poor prognosis could be differentiated.
blood or marrow sample. Dr. Witzig and colleagues studied
According to our experience, there is as yet only one chro-
myeloma cells for production of FGFs and FGFRs. Most of
mosomal region that adds independent prognostic informa-
the cell lines made FGF-2, and some made FGF-5 and FGF-
tion to that already provided by del 13q, ie, presence of a
deletion of chromosome 22q. In the presence of these unfa-
9. The cell lines did not make FGF-1, -3, -4, -6, or -7.
vorable cytogenetic parameters (del 13q and/or del 22q),
There are many other FGFs, but they have not yet been
there were only two additional independent prognostic vari-
studied and less information is known about them.
ables for survival: b2M >4 and immature plasma cell mor-
phology. Again, by combining these unfavorable variables,
Studying the cell lines for FGFRs is also important, because
one can identify distinct groups of patients with significantly
if the myeloma cell makes FGFR and FGF, it is possible that
different survival times.
a myeloma cell can stimulate itself (referred to as autocrine
stimulation). All the cell lines were shown to make at least
It is still unclear which critical gene or genes located on
one FGFR, and synthesis of all four FGFRs was found.
chromosome 13q may be dysregulated and thus contribute to
Myeloma cells were isolated from the bone marrow of eight
such a dismal prognosis. In the introduction to this session,
patients with MM. We found that all samples contained the
Dr. John Shaughnessy reported on the experience at the
Arkansas Cancer Research Center with a set of DNA probes
message for FGF-2 production, one patient's cells had mes-
along the entire 13q arm; two deletion hot spots (one around
sage for FGF-5, and one had FGF-9. Three patients had
the BRCA-2 locus at 13q12 and one at 13q31) were identi-
FGFR-1, and two each had FGFR-2, -3, and 4. Dr. Keith
fied. In further studies using microarray technology, expres-
Stewart of the Princess Margaret Hospital in Canada recent-
sion of 87 genes on chromosome 13 is being investigated;
ly demonstrated that when bone marrow cells that express
these ongoing studies may provide novel insights into the
FGFR-3 were transplanted into mice, the mice developed
molecular pathology of MM with del 13q. In a study present-
cancer of the B cells. This indicates that the abnormal
ed by Dr. Fonseca`s group during the poster session, MM fea-
expression of FGFR-3 on myeloma cells can be oncogenic.
tures were analyzed in the context of chromosome 13 status
In other words, this can cause the cells to become malignant.
in an attempt to define potential mechanisms for the adverse
If a myeloma cell has the t(4;14)(p16;q32), the FGFR-3 is
outcome. However, no clear-cut association with features
placed into a region very important to the function of B-
related to tumor burden, proliferation, or bone marrow
microvessel formation were observed.
cells. This explains one way a PC can become malignant. It
is clear that most cases of MM do not have the
There is an urgent need for effective treatment strategies for
t(4;14)(p16;q32); thus, there appear to be multiple genetic
patients with MM presenting with high-risk cytogenetics. Dr.
pathways to produce MM . It is not clear what the relation-
Shaughnessy reported on conventional cytogenetic and FISH
ship is between the genetic abnormalities and response to
data obtained in the first cohort of patients entered on the
treatment or prognosis. This work indicates that myeloma
Total Therapy II protocol. Patients in whom the del 13q was
cells can make FGFs and FGFRs. What function these
detected by metaphase cytogenetics experienced short event-
cytokines have in myeloma cells is yet to be explained, but
free survival; however, such a negative outcome was not
they may contribute to the increased angiogenesis found in
observed when chromosome 13q was analyzed via FISH. The
the marrow of patients with MM.
biological basis for this observation remains unclear at this
time, and longer follow-up will be needed to reach final con-
clusions. Later in the meeting, Dr. Bart Barlogie, also from
PRIMARY
SECONDARY
the Arkansas Cancer Research Center, reported that del 13q
was also an adverse prognostic feature in the trial with
Timing
Early (initiation) Late (progression)
thalidomide asmonotherapy. However, in a preliminary
analysis of patients being treated with the DT-PACE proto-
B-cell­specific
Usually
No (not active in PCs or
col (dexamethasone, thalidomide, cis-platinum, adriamycin,
mechanism
tumor cells)
cyclophosphamide, etoposide), del 13q did not appear to be
as negative a prognostic factor as in previous analyses.
Ig locus associated Usually
Less often
Conclusions
Present in MGUS
Yes
No
14q32 translocations, which occur in at least 50% of individ-
Heterogeneity in
No
Sometimes
uals with MGUS and in about 75% of patients with MM,
tumor population
appear to be the tumor-initiating event in these cases.
Additional genetic changes then contribute to malignant
Type of karyotypic Simple reciprocal
Usually com-
transformation, although the precise sequence of genetic
plex*
events still needs to be elucidated. As soon as these steps
abnormality
have been initiated, myeloma cells characterized by marked
genetic instability expand. Genetic aberrations occurring at
that level may be critical determinants of prognosis, which is
* Complex = nonreciprocal translocation or insertion, often
particularly evident in the case of del 13q. Further delineation
involving three different chromosomes, inversion, deletion,
of the molecular basis of such high-risk disease as well as its
duplication, or amplification. v
effective treatment remain a challenge in MM research. v
7

---

INNOVATIVE TECHNOLOGIES
Brian Van Ness, Ph.D.
University of Minnesota, Minneapolis, Minnesota
Seilhamer presented evidence that many genes have been
missed, and discovery of rare expressed genes may significant-
ly increase the total gene number. Dr. Reid Asbury of
Amersham-Pharmacia Biotech, Inc. presented new
approaches in protein profiling. Combining two-dimensional
gel electrophoresis and mass spectroscopy, it is now possible
to profile the expression of thousands of proteins in a tissue
sample. Because genetic profiling cannot identify many of
the protein modifications associated with intracellular signal-
ing, protein profiles add significant analysis to tumor biology
and may identify novel targets for therapeutic interventions.
Dr. Asbury presented some initial protein profiles of myelo-
ma cell lines, demonstrating protein changes associated with
INTRODUCTION
interleukin (IL)-6 signaling and ras mutations. Drs. Asbury
and Van Ness are collaborating to identify such proteins in
Understanding of the biology of multiple myeloma (MM) is
myeloma cells and correlate protein profiles with genetic
rapidly being influenced by innovative technologies that are
expression profiles.
focused on defining comprehensive genetic and protein pro-
files of the malignant clone. The session on innovative
The scientific program included a session in which specific
technologies highlighted new applications of gene microarray
applications of these innovative technologies to lymphoid
technologies and the genetic profiling of both myeloma cell-
malignancies were presented. Dr. Louis Staudt, from the
line model systems and patient samples. Integrating new
National Institutes of Health, is one of the leading authori-
technologies into protein profiling is another important
ties on genetic profiling. He presented an overview of the
advance, and the combination of these approaches will likely
technology in which cDNA sequences are spotted on a glass
change the definition of the disease, identify novel therapeu-
slide and hybridized to fluorescent-tagged cDNA generated
tic targets, and influence therapeutic choices.
from RNA samples of lymphoid malignancies. Dr. Staudt
has generated a specific "lymphochip" containing 18,000
HIGHLIGHTS
genes, many of which are selectively expressed in lympho-
cytes along with genes that regulate lymphocyte function,
Although MM is a single, clinically defined disease, there is
including growth factors, receptors, and transcription factors.
considerable genetic heterogeneity among patients that
The goals of Dr. Staudt's genetic profiling of lymphoid malig -
impacts disease progression and response to therapy. Indeed,
nancies were to define genes involved in the pathogenesis of
several years ago, the director of the National Cancer
disease, establish common profiles that may offer novel prog-
Institute issued a challenge to develop new genetic classifica-
nostic indicators, and identify new targets for therapy. Dr.
tions of cancer. In December 2000, a progress-review group,
Staudt demonstrated through the lymphochip microarray
including several members of the myeloma research commu-
analysis of gene expression in diffuse large B-cell lymphoma
nity, identified several focal areas: 1) develop a genetic and
samples that this single diagnosis actually contains two dif-
protein profile of lymphoid malignancies, 2) profile expres-
ferent diseases that differ in expression of hundreds of genes.
sion changes associated with tumor-host interactions, 3)
The two types of lymphoma resemble different types of nor-
from these profiles generate hypotheses that can be tested in
mal B-cell lymphocytes, suggesting that these cancers have
model systems, and 4) use this information to develop novel
distinct cellular origins. Clinically, patients with these two
therapeutic approaches to individual patients. These chal-
types of large B-cell lymphoma had strikingly different
lenges are being met with innovative technologies in gene
responses to chemotherapy. Dr. Staudt presented recently
and protein profiling.
obtained lymphochip profiles of myeloma patient samples
and demonstrated gene expression patterns that distinguish
In a preworkshop symposium, two presentations highlighted
this plasma cell malignancy from other lymphoid malignan-
the technical development and application of genetic and
cies. Expanding this database could provide novel genetic
protein profiling. Dr. Jeff Seilhamer, cofounder of Incyte
classifications and influence therapeutic choices.
Genomics, Inc., one of the leading genomic biotech compa-
nies, presented his views on the human genome project and
Dr. John Shaughnessy from the University of Arkansas pre-
the development of technologies to examine genetic expres-
sented the most comprehensive genetic profile of myeloma
sion profiles, as well as analytical tools that can be used to
patients. Using an Affymetrix oligonucleotide-based chip
query genetic databases. Although recent publications from
containing 6,500 known genes, Dr. Shaughnessy has profiled
both Celera and the public genome project suggested that
more than 100 myeloma patients, as well as a number of nor-
the human genome includes a total of 32,000 genes, Dr.
mal plasma cells. One analytical tool used to evaluate the
8

---

data is the clustering of genes with common expression pat-
and drug-metabolizing genes. There are a number of signifi-
terns. Two major clusters were identified, one containing
cant variations within the normal population of such genes
normal plasma cells, most of the MGUS (monoclonal gam-
that likely influence both risk for developing malignancy and
mopathy of undetermined significance) patient samples, and
response to the highly toxic effects of chemotherapeutic
a group of MM patient samples. Another grouping con-
agents. Dr. Van Ness has been correlating some of the fre-
tained all the myeloma cell lines profiled and two additional
quent genetic polymorphisms with chemotherapy-associated
subgroupings of MM, many with particularly aggressive dis-
toxicities. Preliminary evaluation of the data suggests that
ease. The cluster analysis suggested that gene expression pat-
there may be some forms of the glutathione-S-transferase
terns can discern different subtypes of MM and MGUS. A
gene that correlate with the risk for development of second-
total of 244 genes were identified that were differentially
ary malignacies often associated with cumulative doses of
expressed in normal and malignant plasma cells (137 up reg-
DNA-damaging agents used in therapy. Employing cell line
ulated in MM and 87 down regulated). Genes involved in
model systems to examine gene expression variation, Dr. Van
transcription represented the largest group of altered genes;
Ness has been using the Affymetrix 12,000 gene-detection
genes associated with adhesion, growth control, cell cycle,
platform to examine alterations in gene expression associated
signal transduction, and oncogenesis were also highly repre-
with ras mutations and with myeloma interactions with the
sented. In addition, gene expression patterns correlated
stromal microenvironment. In the collaborative work by Drs.
closely to known chromosomal abnormalities (genes from
Asbury and Van Ness, preliminary data showed that 750
chromosome 13 under represented and genes from chromo-
unique proteins could be separated, with about 15% differen-
some 7 over represented).
tial expression as a result of IL-6 stimulation and about 12%
differential expression resulting from the introduction of a
Dr. Diane Jelinek of the Mayo Clinic has been using a com-
mutant ras gene. Specific identification of these different
mercially available high-density membrane-based microarray
proteins is awaiting mass spectroscopic analysis by Dr. Asbury.
from Research Genetics. This system has required a signifi-
cant effort to address controls and potential artifacts in
In the application of microchip devices, Dr. Christopher
experimental results, and Dr. Jelinek provided a thorough
Backhouse of the University of Alberta presented novel
characterization of approaches to recognize and minimize
applications of microelectronic fabrication technologies to
these artifacts. Using these microarrays, Dr. Jelinek has
produce microchannel networks in glass slides. Within these
focused on analysis of the IL-6 response in cell lines, well
microchannels, reagents can be manipulated by applying
known to provide important growth stimulation in myeloma.
electric fields and can be detected by optical means. These
Interestingly, there are a number of genes she sees upregulat-
microfluidic systems allow the integration of several functions
ed in common with other investigators who profiled patient
on a chip and the analysis of single cells. Recent develop-
samples reported in this session. In addition, Dr. Jelinek is
ments have shown the use of microfluidic chips in the applica-
taking a novel approach to identify a class of important sig-
tion of PCR, electrophoresis sizing, cytometry, and gene-
naling proteins. She has developed a polymerase chain reac-
sequence detection. The signature sequence of the heavy-
tion (PCR)-based technique to examine expression of all
chain immunoglobulin VDJ sequence in myeloma is a clono-
tyrosine kinases, and she presented preliminary anlaysis of
typic marker of the malignancy. In collaboration with Dr.
this class of important signaling proteins in a number of
Linda Pilarski, Dr. Backhouse demonstrated an on-chip
myeloma patient samples. Identification of genes that are
microfluidic analysis of the myeloma clonotypic sequence,
IL-6 responsive in myeloma cells is an important key to
combining PCR cycling on the chip and electrophoretic sepa-
understanding tumor growth, as is identification of the key
ration to distinguish polyclonal products of the normal popula-
downstream tyrosine kinases that are likely to impact atypi-
tion from the monoclonal "spike" produced by the malignant
cal malignant plasma cell proliferation.
clone. Drs. Backhouse and Pilarski also demonstrated a
microchip analysis of RHAMM, an oncogene detected in
Dr. Keith Stewart, from the Toronto General Research
myeloma patients, with various splice variants that may differ-
Institute, has generated a "myeloma chip" analogous to the
entially influence the movement of the malignant cells. This
lymphochip developed by Dr. Staudt. Dr. Stewart's Myeloma
application of microchip technology provides rapid throughput
Gene Database consists of 4,660 nonredundant genes, many
and accurate detection of specific sequences from the malig-
of which appear unique to myeloma cells and represent
nant clone. Such an approach allows analysis of unique prop-
potentially novel genes. His group has classified these
erties of each malignant clone, detection of minimal residual
expressed genes according to putative function. This ongo-
disease, and a rapid analysis of therapeutic interventions.
ing effort is being used to develop disease-specific microar-
rays that may provide the basis for more clearly defining the
It was clear from this session that new technologies are
molecular portrait of myeloma.
quickly redefining important characteristics of the disease
that were not previously available. The comprehensive pro-
Dr. Van Ness presented several approaches to gene and pr o-
filing of gene expression and protein expression, as well as
tein profiling, highlighting the importance of the integration
the rapid analysis of multiple genes that influence disease
of genetic and protein data sets to fully understanding the
progression and therapeutic response, is changing the clinical
functional consequences of gene deregulation in the disease.
picture. The goal is to identify the unique properties and
One area of genetic analysis that should be considered is the
vulnerabilities of each patient's malignant population so that
genetic variation in the population of detoxification genes
a more individualized treatment strategy can be developed. v
9

---

SIGNALING AND MICROENVIRONMENT
Dharminder Chauhan, Ph.D.
Dana-Farber Cancer Institute, Boston, Massachusetts
levels of proteins may sensitize cells to dexamethasone.
Importantly, IL-6's protective effect against dexamethasone
killing also requires growth-promoting proteins, such as
SHP2 (Src-homology protein tyrosine phosphatase-2). SHP2
interacts with RAFTK and stops dexamethasone-induced
cell death. IL-6 also promotes activation of the growth-sig-
naling protein MAPK (mitogen-activated protein kinase).
Dexamethasone activates caspases (enzymes that execute the
death signals). In contrast, IL-6 increases levels of proteins
such as IAPs (inhibitors of apoptosis proteins), which block
caspase functions and thereby prevent dexamethasone-
induced cell death. Interestingly, higher levels of anti-cell-
INTRODUCTION
death proteins such as IAPs are observed in patients with
advanced myeloma. New treatment strategies based on
Most anticancer drugs kill cancer cells through a process
either inhibiting the survival proteins or enhancing the func-
called programmed cell death, or apoptosis. Disruption of the
tionality of death proteins may improve drug efficacy.
apoptotic process by factors (cytokines) present in the tumor
microenvironment may promote tumor initiation, progres-
Dr. Jernberg Wilkund of Uppsala University in Sweden dis -
sion, and treatment resistance. This session focused on iden-
cussed the roles of interferon-gamma (IFN-) and insulin
tifying various signaling proteins that can be targeted for
growth factor (IGF-I), which may determine the sensitivity
therapeutics based on their ability to either trigger apoptosis
of MM cells to dexamethasone or Fas-induced cell death.
or inhibit growth/survival of tumor cells.
Blocking the IGF-I Receptor (IGF-IR) with anti-IGF-IR
antibodies increases dexamethasone- and Fas-induced death
HIGHLIGHTS
in MM cells. Using antibodies specific to either IFN- or
IGF-IR can restore the normal cell-death pathway. IGF-IR
In myeloma, tumor cells are predominantly localized to the
signaling may confer resistance to apoptosis and may consti-
bone marrow microenvironment because they adhere to
tute a novel mechanism for the development of chemoresis-
bone marrow cells. This interaction between tumor cells and
tance in MM.
bone marrow cells stimulates production of various cytokines,
such as interleukin (IL)-6, which not only potentiate growth
Dr. Amiot of the Institut de Biologie in France discussed
and survival of multiple myeloma (MM) cells but also sup-
another growth-signaling pathway that is triggered by IL-6
port development of drug resistance. High serum levels of
via a protein called protein kinase C-delta (PKC-).
IL-6 observed in MM patients contribute to chemoresistance
Rottlerin, a specific inhibitor of PKC-, induces death in
and treatment failure. For example, dexamethasone, a drug
myeloma cells. Additional proteins that regulate the PKC
used in treatment of MM, kills MM cells; however, in the
pathway are PI3K/AKT and MAPK. Treatment of myeloma
presence of IL-6, the effectiveness of dexamethasone is sig-
cells with wortmanin, a specific blocker of PI3K, stops the
nificantly reduced. Thus, the cytokines present within the
PKC--induced growth signals. Targeting PKC- and associ-
bone marrow microenvironment may negatively regulate
ated proteins may therefore be useful therapeutic strategies.
drug-induced death in MM cells. Defining the signaling
pathways mediating cell death, growth, and chemoresistance
Dr. Regis Bataille, also of the Institut de Biologie, discussed
in MM cells may lead to new and effective treatment strate-
IL-6 as an essential factor for the expansion/growth of MM
gies based on targeting growth and survival proteins.
cells within the bone marrow microenvironment. Based on
these studies, numerousnew therapeutic approaches were sug-
Dr. Chauhan discussed the interactions between growth­ and
gested, including neutralizing the IL-6 receptor present on
cell-death­signaling pathways in myeloma cells. In particu-
the MM cell surface and using anti-IL-6 antibodies. Several
lar, dexamethasone-mediated myeloma cell killing requires
clinical trials are currently under way using neutralizing anti-
enzymatic activation of proteins such as RAFTK (related
IL-6 antibody in combination with conventional drugs such
adhesion focal tyrosine kinase), Smac (second mitochondrial
as, dexamethasone and melphalan. These studies have
activator of caspases), and caspase-9 Deleting these proteins
shown encouraging results, with remissions in advanced-stage
renders cells resistant to dexamethasone; conversely, higher
MM patients. IL-6 also induces the anti-cell-death protein
10

---

MCL-1, which may be a useful therapeutic tool to stop
tumor cell growth and survival in the bone marrow.
Another mechanism whereby the bone marrow microenvi-
ronment affects myeloma cell growth was discussed by Dr.
Vanderkerken of the Free University in Belgium. A distinct
category of factors (chemokines) are produced by bone mar-
row cells, which attract MM cells and thereby facilitate hom-
ing of MM cells to the bone marrow. For example, MCP-1
(monocyte-chemoattractant protein-1) or IGF-I is secreted
by bone marrow cells and binds to receptors present on
myeloma cells (CCR2) and IGF-IR. Importantly, this inter -
action can be prevented by using specific antibodies to
MCP-1 or IGF-I and thereby blocking homing of MM cells
to bone marrow. Other molecules that were shown to play
similar roles include MMP-9 and CD44v6. v
11

---

ANGIOGENESIS IN MULTIPLE MYELOMA
Ivan Van Riet, Ph.D.
Free University Brussels, Brussels, Belgium
myeloma cells within the bone marrow compartment is asso-
ciated with increased activity of endothelial cells, resulting
in neovascularization. Dr. Vincent Rajkumar of the Mayo
Clinic illustrated that microvessel density (MVD) in the
bone marrow progressively increases from monoclonal gam-
mopathy of undetermined significance to smoldering MM to
MM and that angiogenesis has a prognostic value in MM.
Superior survival for MM patients with low MVD in the
bone marrow was also reported by Dr. Orhan Sezer of
University Hospital Charite. He noted that bone marrow
MVD significantly decreases in MM patients who achieve
remission regardless of the type of chemotherapy they
received. This decrease seems to be associated with pro-
INTRODUCTION
longed progression-free survival. Dr. James Berenson of
Cedars-Sinai Cancer Center mentioned that relapse after stem
Angiogenesis plays an important role in the pathogenesis
cell transplantation is associated with an increase in MVD.
and progression of multiple myeloma (MM). Recent research
identified myeloma cell-secreted factors (such as vascular
Molecular aspects of angiogenesis in MM
endotherlial cell-growth factor [VEGF] and [bFGF]) that
have the potential to trigger this process. Inhibition of neo-
Although the role of angiogenesis in the growth and metas-
vascularization by thalidomide or its analogues
tasis of solid tumors has been clearly established, less is
(immunomodulatory drugs, or IMiDs) seems to be a promis-
known about the underlying mechanisms that mediate this
ing therapeutic approach for MM. In addition, the mecha-
process in MM. Several presentations focused on the role of
nisms of action of novel therapeutic molecules in MM (such
specific angiogenic molecules in the pathogenesis of MM.
as the proteasome inhibitor PS-341 and zoledronic acid)
Both Dr. Rajkumar and Dr. Bellamy of the University of
seem to be partially based on their antiangiogenic activity.
Arizona reported on the expression of VEGF by myeloma
Targeting angiogenesis may become an important aspect of
cell lines and myeloma plasma cells in the majority of the
disease control in MM.
patients tested. Dr. Berenson demonstrated the existences of
a correlation between microvessel density, VEGF expression
HIGHLIGHTS
by plasma cells, and the number of plasma cells in myeloma
bone marrow samples. Dr. Kenneth Anderson of the Dana-
Angiogenesis, or neovascularization, is the formation of new
Farber Cancer Center mentioned that VEGF might not only
blood vessels from preexisting vessels. This process occurs in
be important for the formation of new blood vessels in MM
multiple steps and is triggered by positive angiogenic factors
but also has the potential to stimulate directly the prolifera-
released by the tumor cells or induced in the microenviron-
tion and migration of myeloma cells. His group demonstrat-
ment of the tumor cells. These factors stimulate the migra-
ed that production of VEGF by myeloma cells can be stimu-
tion and proliferation of endothelial cells, resulting in the
lated by activation of the CD40 molecule on the surface of
formation of new capillaries. Successful neovascularization is
the tumor cells. One study revealed that high levels of VEGF
believed to be critical for growth and metastasis of solid
in the serum of MM patients is associated with extramedular
tumors. Recently it became clear that neovascularisation is
growth of tumor cells.
also part of the pathogenesis of hematologic malignancies,
including MM.
Dr. Thomas Witzig of the Mayo Clinic reported on the
expression of another potential angiogenic molecule in MM,
Angiogenesis is involved in the pathogenesis of MM.
fibroblast growth factor (FGF). He found that myeloma cell
MM cells have the capacity to communicate with different
lines express different FGF types (FGF-2, -5, and -9) and also
cell types in their natural microenvironment, the bone mar-
FGF receptors (FGFRs), indicating the possible involvement
row. Molecular interactions with stromal fibroblasts sustain
of an autocrine FGF/FGFR loop in the growth control of
the survival and growth of the tumor cells, and myeloma
myeloma cells. Dr. Van Riet illustrated that human myeloma
cell-mediated activation of osteoclasts results in the induc-
cell lines can directly trigger the neovascularization process
tion of osteolytic lesions. More recently it became evident
by stimulating the migration and proliferation of human
that myeloma cells also interact with endothelial cells.
endothelial cells. He demonstrated that although VEGF-A
Interaction between endothelial cells and myeloma cells that
and bFGF are both functionally involved in these steps, the
circulate in the blood can result in the migration and subse-
major effect is caused by VEGF-A. It was found that the
quent homing of the tumor cells to the bone marrow. Several
expression of both angiogenic factors is triggered by molecu-
recent clinical observations indicate that the presence of
lar interactions between myeloma cells and bone marrow
stromal cells. He also showed that myeloma cell lines express
12

---

additional angiogenesis-promoting molecules such as VEGF-
alone. Dr. Durie mentioned that the combination of thalido-
B and angiopoietin-1; however, their functional role in MM
mide with Biaxin or Biaxin/dexamethosone can be active for
remains to be determined. In one study, serum levels of dif-
failing patients.
ferent angiogenic factors (VEGF, bFGF, and hepatocyte
growth factor (HGF) were measured in a group of MM
Dr. Donna Weber of M.D. Anderson Cancer Center demon-
patients before and after treatment. It was found that MM is
strated that in a group of 44 previously untreated MM
associated with an increase in serum bFGF levels in advanced
patients, the response rate with thalidomide as monotherapy
disease and that effective chemotherapy is associated with a
was approximately one half (36%) of that with the thalido-
significant decrease in the levels of bFGF, VEGF, and HGF.
mide/dexamethosone combination (69%). The promising
activity of thalidomide/dexamethosone in previously untreated
Dr. Joshua Epstein of the University of Arkansas presented the
patients with active MM was also reported by Dr. Rajkumar.
SCID-hu model, in which human myeloma cells are injected
in human fetal bones that are implanted in severe combined
Some novel drugs were discussed that not only target the MM
immunodeficiency disease (SCID) mice. In this model, myelo-
cell but also act against the microenvironment, including the
ma cells not only grow in human bone at the site of injection
formation of new blood vessels. Several novel proteins were
but can also migrate and home to another bone implanted in
recently isolated that inhibit angiogenesis including VEGF
the same animal. Interestingly, recombinant endostatin (a nat-
inhibitor (VEGFI). Previous studies demonstrated that
ural antiangiogenic factor) administered to tumor-bearing
VEGFI inhibits angiogenesis and may directly induce apopto-
SCID-hu hosts had a clear antimyeloma effect that was associ-
sis of tumor cells without inhibiting normal cells. Dr.
ated with a strong inhibition of microvessel formation. Similar
Berenson demonstrated that VEGFI also directly affects the
effects were seen with adenovirus endostatin gene therapy. It
growth of human myeloma cell lines. He showed that bispho-
seems that survival and growth of myeloma cells in this model
sphonates, such as zoledronic acid, not only inhibit the devel-
require active neovascularization.
opment of osteoclasts but also seem to be markedly antiangio-
genic, possibly by affecting the production of VEGF.
Angiogenesis as a therpeutic target in MM
Dr. Anderson gave an overview of the different biological
Several presentations illustrated the therapeutic potential of
effects of the proteasome inhibitor PS-341 on myeloma cells.
thalidomide in the treatment of MM patients. Dr. Anderson
This molecule directly inhibits the growth of myeloma cells,
summarized the different biological effects of thalidomide
overcomes resistance to apoptosis in MM cells conferred by
that contribute to its therapeutic benefit in MM.
interleukin-6, and reduces the adhesion of tumor cells to
Thalidomide not only has antiangiogenic activity but also
bone marrow stroma. This impairment of binding to bone
directly induces apoptosis, or growth ar rest, of myeloma cells.
marrow stromal cells will also reduce VEGF secretion, lead-
The major angiogenesis-inhibiting effect of thalidomide
ing to inhibition of angiogenesis. An ongoing clinical trial
relates to its potential to abrogate the secretion of VEGF.
should explore the role of PS-341 in the treatment of MM.
Moreover, thalidomide stimulates the patient's immune sys -
Dr. Anderson's group also demonstrated that IMiD derivates
tem to kill tumor cells. Dr. Bart Barlogie of the Arkansas
of thalidomide have significantly more potent effects against
Cancer Research Center mentioned that thalidomide can
myeloma cells than does thalidomide itself. These include
also down regulate c-myc, an oncogene that is activated in
inhibition of VEGF-induced proliferation and migration of
the tumor cells of some MM patients.
myeloma cells and antiangiogenic activity. A Phase I study
to test the safety and efficacy of the IMiD CC5013 for treat-
Several studies indicated that thalidomide is an effective
ment of MM patients was recently started.
treatment for patients with advanced MM. Dr. Barlogie
reported on the effect of thalidomide treatment in a group of
The biological and clinical importance of angiogenesis in the
169 patients with advanced and refractory disease, resulting
pathogenesis and treatment of MM was addressed by numer-
in event-free survival and overall of 60% and 20%, respec-
ous speakers. The increased density of blood vessels in the
tively. Dr. Rajkumar confirmed that thalidomide treatment is
bone marrow of MM patients not only is associated with the
effective in 25% to 35% of patients with refractory disease.
proliferation of plasma cells but seems also to correlate with
Similar results were reported by several other groups. In most
the prognosis for the disease. Myeloma cells secrete mole-
studies, daily doses of thalidomide between 200 and 800 mg
cules that have the potential to trigger formation of new
have been used. Because of the considerable toxicity, Dr.
blood vessels in the bone marrow. The production of these
Brian Durie of Cedars-Sinai Cancer Center evaluated less
factors seems to be regulated by molecular interactions
toxic doses (50 to 400 mg) and observed clinical responses in
between the tumor cells and the host bone marrow microen-
44% of patients with relapsing MM.
vironment Angiogenesis may be a promising therapeutic tar-
get in MM. Treatment with thalidomide has marked activity
Several studies evaluated the clinical effect of treatment with
in MM patients, even in those with refractory disease.
thalidomide in combination with other therapeutic agents. It
Ongoing studies need to clarify the role of thalidomide in
was shown that thalidomide plus dexamethosone was highly
combination with other therapeutic agents as well as its up-
effective for patients with relapsed or refractory MM.
front use. A new series of therapeutic molecules act against
Another study confirmed these data but also revealed that
the myeloma cell and its microenvironment, including the
the thalidomide/dexamethosone combination does not
formation of new blood vessels. These molecules either are
lengthen remission duration compared with thalidomide
being or soon will be evaluated in clinical trials. v
13

---

TREATMENT MODALITIES AND SYMPTOM MANAGEMENT
Robert A. Kyle, M.D.
Mayo Clinic, Rochester, Minnesota
Miguel of the University of Salamanca in Spain reported on
efforts to establish an international prognostic index for MM.
They noted that the Durie-Salmon Staging System has been
a standard for a quarter century, but it has many shortcom-
ings. Newer prognostic factors are available, but there is no
agreement on the best system. Prognostic factors were dis-
cussed at the International Myeloma Foundation retreat and
at the American Society of Hematology Meeting in 2000.
Criteria for treatment were developed, and Dr. Kyle present-
ed the criteria at the International Prognostic Group
Meeting in Banff.
Therapy
INTRODUCTION
If the patient is less than 70 years old, the physician should
seriously consider an autologous peripheral blood SCT. The
Multiple myeloma (MM) must be differentiated from mono-
stem cells must be collected before the patient is exposed to
clonal gammopathy of undetermined significance (MGUS),
alkylating agents. The two major shortcomings of autologous
smoldering multiple myeloma (SMM), and primary amyloi-
SCT are that myeloma is not eradicated even with large doses
dosis. Treatment is not recommended unless symptoms
of chemotherapy, total body irradiation (TBI), or both and
develop or abnormalities progress. If the patient is less than
that autologous peripheral stem cells are contaminated by
70 years old, one should consider autologous peripheral stem
myeloma cells or their precursors. The mortality rate with
cell transplant (SCT). Mortality is low, but this procedure is
autologous SCT is only 1% to 2%. One can proceed with the
not curative, and most patients relapse. There is no con-
transplant as soon as the patient has recovered from stem cell
vincing evidence that double or tandem autologous SCTs are
collection, or, following collection, one can treat the patient
superior to single SCTs. Mortality with allogeneic bone mar-
with alkylating agents and delay the transplant until progres-
row transplantation (BMT) is high, so "mini-allogeneic"
sive disease develops. Survival is the same with both
transplants are being developed.
approaches, but patients given early transplants are spared the
HIGHLIGHTS
inconvenience and cost of chemotherapy Currently autolo-
gous SCT is applicable for up to 50% of patients with MM.
Minimal criteria for the diagnosis of MM include bone mar-
row with more than 10% plasma cells or tissue biopsy speci-
In a prospective study comparing melphalan plus TBI with
men demonstrating monoclonal plasmacytosis and at least
melphalan alone, the response rate, event-free survival
one of the following: monoclonal protein (M-protein) in the
(EFS), and overall survival (OS) were not different with the
serum (usually >3 g/dL), M-protein in the urine, or lytic
two regimens. The toxicity of melphalan 200 mg/m2
lesions. MGUS, SMM, and primary amyloidosis must be dif-
(MEL200) was significantly lower than that of melphalan
ferentiated from MM. Patients with MGUS or SMM should
plus TBI. In an effort to improve the preparative regimen,
be identified and not treated unless symptoms develop or
studies are being conducted with Holium-166-DOTMP or
laboratory abnormalities progress, because they may remain
Samarium-153-EDTMP in conjunction with melphalan.
stable for many years. The size of the serum M-protein,
Alfa2 interferon (2-IFN) prolonged initial progression-free
amount of M-protein in the urine, and number of plasma
survival, but after longer follow-up there was no difference in
cells in the bone marrow are helpful in differentiating
survival. Idiotype-treated dendritic cells have been used to
between benign and malignant plasma cell proliferative dis-
prolong the duration of response.
orders. The plasma cell labeling index (measurement of the
synthesis of DNA by plasma cells) is useful. If this index
The selection of CD34 stem cells might be beneficial,
increases, the patient has active MM or will soon have symp-
because this results in a 2- to 3-log reduction in tumor cells.
tomatic disease. On the other hand, 40% of patients with
However, EFS and OS are not significantly different between
MM have normal labeling indices. Increased numbers of cir-
patients with or without stem cell selection. At present,
culating plasma cells of the same isotype are present in 80%
eradication of myeloma is the more important goal, but as
of patients with active MM, but there are few or no circulat-
preparative regimens improve, contamination of the rein-
ing plasma cells in patients with MGUS or SMM. The
fused cells will assume greater importance.
patient must be followed with measurements of M-protein in
the serum and urine and periodic evaluation of clinical and
Dr. Mario Boccadoro and colleagues of the University of
laboratory parameters.
Torino reported the results of studies using intermediate
doses of melphalan. They treated 71 MM patients with two
Prognosis
or three courses of melphalan 100 mg/m2 (MEL100) followed
by stem cell support. The median patient age was 64 years.
Drs. Philip R. Greipp of the Mayo Clinic and Jesus San
14

---

The patients were matched on the basis of age and beta2-
intensive arm. Survival data will be forthcoming.
microglobulin (2-MG) levels with patients treated with oral
melphalan and prednisone (MP). Complete remission (CR)
Double or tandem autologous SCT has been recommended,
occurred in 47% of the MEL100 and 5% of the MP patients.
but in a randomized trial by the French Myeloma Group com-
Median EFS was 34 months in the MEL100 group and 17.7
paring single and double transplant, 2-year EFS and OS were
months in the MP group (P <0.001). Median OS was 56+
not different. In a subsequent analysis, patients with double
months for MEL100 and 48 months for MP (P<0.01). In a
transplants at 5 years post-diagnosis had a survival rate of
multivariate analysis, superior EFS and OS were observed in
85% compared with 70% for those with single transplants.
patients presenting with low 2-MGlevels at diagnosis and in
patients receiving MEL100.
Allogeneic BMT has the advantage that the graft contains no
tumor cells and may produce a graft-vs-myeloma effect.
In a second analysis, Dr. Boccodoro compared the effect of
However, only 5% to 10% of patients with MM are candi-
MEL100 with that of standard MP in patients more than 65
dates for conventional allogeneic BMT from HLA-matched
years of age. Thirty-one patients aged 65 to 77 years (medi-
donors. The mortality rate is 20%-30%. In an effort to
an 67) were treated with two or three courses of MEL100
reduce mortality and increase the graft-vs-myeloma effect,
and compared with a comparable group of 31 patients treated
investigators are using "mini-allogeneic" BMT with a non-
with MP. The patients were matched according to their
myeloablative regimen consisting of melphalan or fludarabine
stage at diagnosis and their serum 2-MGlevels. The EFS
plus melphalan. The depletion of T cells may also decrease
(P<0.005) and OS (P<0.01) were superior for the MEL100
mortality. The use of donor peripheral blood mononuclear
patients to those for the patients receiving MP. Thus,
cells after allogeneic BMT often produces benefit. these
MEL100 appeared to be superior to standard MP in both the
efforts are both important avenues of investigation.
general population and in patients more than 65 years of age.
Systemic chemotherapy is needed for patients older than 70
The investigators next studied MEL100 versus MEL200.
years who have symptomatic MM or younger persons for
They compared 81 patients treated with MEL100 with 81
whom transplantation is not feasible. The most commonly
patients receiving MEL200 matched for 2-MG and stage.
used regimen is MP. The melphalan dosage must be altered
These patients were treated with single (n=45) or double
so that there is some cytopenia 3 weeks after therapy is
(n=36) autologous transplantation. Treatment-related mor-
begun. MP produces an objective response in 50% to 60% of
tality was 4% with MEL100 and 7% with MEL200. The CR
patients. Combinations of alkylating agents produce a better
rate was 43% with MEL100 and 63% with MEL200. EFS
response rate but no significant difference in survival.
was 30 months in the MEL100 group and 33 months in the
Chemotherapy should be continued until the patient reaches
MEL200 group. OS was 57 months for MEL100 and 53
a plateau state. Benefits of maintenance therapy with 2-
months for MEL200. The investigators concluded that
IFN following conventional chemotherapy is controversial.
MEL100 was similar to MEL200 from the standpoint of CR,
In a large meta-analysis, there were modest response duration
EFS, and OS despite a significant patient age difference (63
and survival benefits with 2-IFN in both induction and
vs. 50 years) (P<0.0001Although the CR rate improved by
maintenance regimens.
20% with MEL200, this was not enough to produce a signifi-
cant outcome improvement. One should exercise caution
Dr. Joan Bladé of the Hospital Clinic, Hematology in
when drawing conclusions from these nonrandomized trials.
Barcelona pointed out that renal insufficiency is present at
diagnosis in 20% of patients with MM, and one-third of
Dr. J. Anthony Child and colleagues of the Medical
them die within the first 2 months of therapy. Renal insuffi-
Research Council Trials in the United Kingdom reported
ciency is reversible in up to 50% of patients, particularly
their experience with standard ABCM chemotherapy versus
when its degree is moderate and it is related to precipitating
intensive therapy with high-dose melphalan and stem cell
factors such as hypercalcemia or dehydration. Factors associ-
support. Two hundred and three patients were randomized
ated with renal function recovery are serum creatinine level
to ABCM (doxorubicin 30 mg/m2 IV and BCNU 30 mg/m2
and amount of proteinuria as well as hypercalcemia. Patients
IV, day 1; melphalan 6 mg/m2/day PO and cyclophosphamide
who recover their renal function have a survival rate similar
100mg/m2/day PO, days 22-25), with cycles repeated every 6
to that of patients with no renal failure at diagnosis. The
weeks until a plateau was achieved, and 204 were random-
response of renal failure to chemotherapy is approximately
ized to C-VAMP (doxorubicin 9 mg/m2/day and vincristine
40%. Response to therapy and severity of renal failure are
0.4 mg/day as a mixed continuous infusion, days 1-4; methyl-
factors significantly associated with survival. The treatment
prednisolone 1 g/m2 [max 1.5 g] IV or PO, days 1-5;
of choice for patients with creatinine >4.0 mg/dL with nono-
cyclophosphamide 500 mg IV bolus, days 1, 8 and 15), with
liguric renal failure is VAD or dexamethasone along with
cycles repeated every 3 weeks to a maximum response. All
early plasma exchange. However, plasma exchange is unlike-
C-VAMP patients except those showing disease progression
ly to be beneficial got patients with advanced myeloma cast
went on to receive MEL200 with peripheral stem cell auto-
formation and renal failure severe enough to necessitate dial-
graft. All patients in both regimens were offered mainte-
ysis. Long-term hemodialysis or peritoneal dialysis is worth-
nance therapy with -IFN at 3 megaunits SC 3x/week esca-
while for patients with MM and end-stage renal failure.
lating to 6 megaunits, if tolerated, until relapse. The majori-
ty of patients had bone pain (72%) and multiple lytic lesions
Almost all patients who respond to chemotherapy eventually
(53%). The serum 2-MGvalue at presentation was 4.1
mg/L in patients in the standard arm and 4.2 mg/L in the
Please see page 17
15

---

BONE METABOLISM, BONE DISEASE, AND THE BISPHOSPHONATES
Gregory R. Mundy, M.D.
University of Texas Health Science Center, San Antonio, Texas
inhibit this enzyme apparently correlates very closely with
their potency and efficacy as antiresorbtion agents. The most
potent of the antiresorptive bisphosphonates are zoledronic
acid and risedronate. Dr. Pearse's work suggests that these
compounds then inhibit a chemical modification of small
membrane-bound proteins (GTPase proteins); this is known as
prenylation. This in turn has a number of serious conse-
quences for the cell that ultimately lead to its death by apop-
tosis and loss of its capacity to stimulate bone resorption. Dr.
Pearse and his colleagues have found that similar changes may
also occur in myeloma cells and suggest that part of the antitu-
mor effect of the bisphosphonates may be related to this
capacity to cause tumor cell apoptosis. Dr. Green reviewed
other in vitro effects of the bisphosphonates that could be
INTRODUCTION
linked to their capacity to cause tumor cell apoptosis, includ-
There is clearly an increasing awareness within the myeloma
ing decreased release of interleukin-6 by osteoclasts, myeloma
community of the effects of myeloma on the skeleton. At
cells, and osteoblasts; effects on cell proliferation; effects on
the 8th International Myeloma Workshop, there was a full
tumor cell apoptosis; and effects on angiogenesis. These
session devoted to this topic, and one of the preworkshop
effects may be enhanced by other agents such as dexametha-
symposia dealt with bone-active drugs in the bone microen-
sone and chemotherapeutic agents such as Taxol.
vironment. Attendance at these sessions was excellent, and
there is obvious interest in this area.
The major issue is whether these anti-tumor effects have rel-
evance to the use of bisphosphonates in vivo. Although
A major impetus for the increased interest in bone-active
most of them occur at quite large concentrations of the bis-
drugs has been the advent of bisphosphonates, which were
phosphonates in in vitro experiments, it is possible that these
approved for use in myeloma bone disease by the Food and
concentrations may be achieved in vivo, since bisphospho-
Drug Administration (FDA) 6 years ago. This session focused
nates are concentrated in the bone microenvironment by
on the current status of bisphosphonates, their potential as
their capacity to bind to calcified matrices. However, in
direct antitumor agents, their effects on tumor burden, the
most of the preclinical models of myeloma in which bisphos-
molecular targets for bisphosphonates in osteoclasts and
phonates have been tested so far, the bisphosphonates do not
myeloma cells, and consideration of newly described mediators
have major effects on myeloma cell apoptosis or myeloma
of osteoclastic bone resorption whose production may be
cell death, whereas they are very effective inhibitors of bone
excessive in myeloma, namely RANK ligand and macrophage-
resorption. This is an important area that clearly needs fur-
inflammatory protein. There was also discussion of the poten-
ther investigation. One of the issues may be that there are a
tial utility of new therapeutic agents that interfere with the
number of different animal models of myeloma including the
cell-cell interactions responsible for bone-resorbing activity in
5T models as well as the human severe combined immunod-
myeloma and particularly of the specific inhibitors of RANK
eficiency disease (SCID) models.
ligand, namely RANK.Fc and osteoprotegerin, and the use of
antagonists to macrophage-inflammatory protein. The poten-
Dr. Babatunde O. Oyajobi of the University of Texas Health
tial of markers of bone resorption for staging disease and
Science Center at San Antonio discussed some of the newly
assessing prognostic risk was addressed as well.
described mediators responsible for bone destruction in
myeloma, with particular emphasis on RANK ligand. He has
HIGHLIGHTS
found that myeloma cells when cocultured with bone marrow
stromal cells increase expression of RANK ligand, which has
Dr. Jonathan Green of Novartis Oncology reviewed the struc-
the capacity to stimulate osteoclast formation and activity.
ture and properties of the bisphosphonates in current clinical
use and discussed in some detail their potential as antitumor
To determine the significance of RANK ligand and osteo-
agents. The bisphosphonates have now achieved widespread
clastic bone resorption, he used an antagonist to RANK lig-
use, with a total market of more than $1.5 billion, more than
and known as RANK.Fc, which comprises a genetically engi-
one third of which is for cancer-related indications. This
neered hybrid molecule consisting of the extracellular
rather simple group of compounds falls into two classes, one of
domain of RANK linked to a fragment of immunoglobulin
which contains nitrogen. The nitrogen-containing bisphos-
(Ig). This soluble chimeric protein then acts as a functional
phonates, which by and large comprise the newer bisphospho-
antagonist to RANK ligand, binding to its cognate-receptor
nates, have a different molecular mechanism of action from
rank. He used this in the 5T murine model of myeloma bone
that of the older compounds etidronate and clodronate. This
disease and found very effective inhibition of bone resorption,
was discussed in detail by Dr. Roger Pearse of Cornell
similar to what is seen in this model with the powerful bis-
University Medical College. These bisphosphonates inhibit
phosphonates. However, he also observed a marked reduc-
an enzyme in the mevalonate pathway of cholesterol biosyn-
tion in tumor burden reflected by a decrease in the tumor vol-
thesis. The major enzyme targeted appears to be farnesyl
ume in bone and in the circulating concentrations of IgG2b,
diphosphate synthase, and the capacity of these compounds to
the monoclonal protein produced by myeloma cells that
16

---

serves as a marker of tumor burden in this model.
Treatment Modalities and Symptom Management -
Continued
Dr. Pearse found very similar results. He observed enhanced
expression of RANK ligand (also called TRANCE) by myelo-
relapse if they do not die of another disease. VAD has been
ma cells and stromal cells when they were cocultured. He also
the most effective regimen, but thalidomide produces an
found that RANK.Fc caused marked inhibition of bone
objective response in about one third of patients with refracto-
destruction induced by myeloma in two murine models. He
ry disease. The addition of dexamethasone to thalidomide
used the model in which SCID immunocompromised mice are
may improve the response rate. Other new agents include the
irradiated and inoculated with human fetal bone and human
myeloma cells. In these circumstances he saw a marked reduc-
immunomodulatory drug CDC-501, an analogue of thalido-
tion in bone lesions and tumor burden. He also noted that
mide. In addition, 2-methoxy estradiol, farnesyl transferase
RANK.Fc prevented the development of paralysis in the hind
inhibitors, and the proteosome inhibitor PS-341 are all poten-
limbs of the tumor-bearing mice and blocked the progression of
tial agents for therapy. The challenge is to develop new and
multiple myeloma; he concluded that RANK ligand deregula-
better agents to prolong survival and ultimately cure MM.
tion may be responsible for tumor progression. In addition, he
noted that in myeloma marrow there was decreased expression
Dr. Finn Wisløff of the Nordic Myeloma Study Group empha-
of the soluble decoy receptor for RANK ligand, osteoprotegrin.
sized the importance of quality of life (QOL) for patients with
MM. He reported on the experience of the Group`s experi-
A number of posters also addressed this issue. They included
ence, which represents more than 100 hospitals in Denmark,
work by Penser, Anderson, Barsley, Guiliani, Abe, and Croucher.
Norway and Sweden. This comprises a population of approxi-
There are some differences in the findings from the different
mately 15 million people. The group used the European
groups, possibly related to differences in the antibodies used to
Organization for Research and Treatment of Cancer question-
detect RANK ligand, the cell lines used, and the animal mod-
naire. It was completed prior to treatment and at intervals
els. However, all investigators agree that there is increased and
throughout the course of the disease by more than 80% of the
deregulated expression of RANK ligand in myeloma, this
patients. Quality of life was compared to reference data
expression is enhanced by cell-cell contact and co-culture of
obtained from the Norwegian population adjusted for age and
myeloma cells with stromal cells, and RANK.Fc is a very
gender. They also obtained data on cost of therapy.
effective inhibitor of osteoclast stimulation and possibly of
bone resorption. Open issues include the importance of down
At diagnosis, myeloma patients have a substantial reduction in
regulation of OPG, the relative importance of the myeloma
QOL. There is a large decrease in physical and role function-
cell as a source of RANK ligand, and whether OPG has simi-
ing and a moderate impairment of social and emotional func-
lar effects to those of RANK.Fc in decreasing tumor burden.
tion. Pain and fatigue are substantial problems. Patients who
Dr. Gregory Mundy presented data from Dr. David Roodman
achieved objective response with MP experience substantial
focusing on the role of macrophage-inflammatory protein in
improvement in QOL. Those who had stabilization of their
the osteolysis associated with myeloma. Investigators found
disease even without objective response also had improved
that a biologically active mediator of osteoclast formation in a
QOL. The pain scores were substantially reduced and
cDNA expression library from a patient with myeloma was
approached those of the reference population. The cost of MP
detected and characterized to be a macrophage-inflammatory
was approximately $10,000. Dr. Wisløff concluded that oral
protein. Dr. Roodman found that bone marrow plasma super-
treatment with MP prolongs survival by approximately 2 years,
natants from patients with myeloma had powerful osteoclast-
with good QOL. Consequently, it was considered cost-effective.
stimulating activity that increased osteoclast formation from
human marrow cultures and whose production correlated with
The addition of a-IFN to MP during induction and mainte-
severity of disease. When this soluble factor was incubated
nance therapy produced a 5- to 6-month prolongation of the
with antibodies to macrophage-inflammatory protein, the
plateau state and a 3-month OS increase. Patients receiving
bone-destructive lesions were totally inhibited. The human
a-IFN had a significant reduction in QOL during the first
ARH-77 cell line was stably transfected with antisense con-
year of treatment. There was no improvement in QOL asso-
structs to macrophage-inflammatory protein and then inoculat-
ciated with the plateau phase. The cost was estimated at
ed into irradiated SCID mice. There was a marked reduction
$110,000 per year, which was considered unacceptably high.
in associated osteolysis and development of bone lesions.
Tumor burden was also reduced. This work raises the possibili-
In a population-based study, the survival advantage of high-
ty that macrophage-inflammatory protein may be an important
dose melphalan with autologous stem cell support was com-
mediator of the osteoclast stimulation that occurs in myeloma.
pared with that of conventional chemotherapy in newly
diagnosed symptomatic myeloma patients less than 60 years
Finally, Dr. Facon presented an evaluation of markers for bone
resorption in patients with monoclonal gammopathy of unde-
of age. During the first 6 months, the patients in the high-
termined significance and multiple myeloma. These markers
dose group had significantly lower scores for QOL and for
are thought to reflect the rate and extent of bone resorption
social functioning. At 12 and 24 months, QOL was similar
and the activity of bone-resorbing and bone-forming cells. The
in the high-dose and conventional chemotherapy groups,
report indicated that three markers of bone resorption, namely
and at 36 months, there was a trend toward less fatigue and
deoxypyridinylene, NTX and CTX gave useful information.
pain in the high-dose group. Thus, the 18 months of pro-
These urinary markers correlated very well, and the investiga-
longed survival appeared to be associated with good QOL.
tors recommended that they play an important role in the stag-
The cost relative to conventional therapy with MP was esti-
ing of the disease and may provide extremely useful informa-
mated at $27,000, which was considered acceptable. v
tion when a patient is undergoing prognostic assessment. v
17

---

ALLOGENEIC AND AUTOLOGOUS TRANSPLANTATION
Jesus San Miguel, M.D.
University of Salamanca, Salamanca, Spain
allogeneic.
A total of 320 patients were enrolled in the Italian study
("Bologna 96"). The randomization was one transplant with
melphalan (200 mg/m2) versus two transplants (the first with
the same regimen and the second with melphalan 120 mg/m2
plus Busulfan 12 mg/kg). The results failed to show signifi-
cant differences in CR and OS rates. In contrast, patients
receiving two transplants enjoyed longer EFS.
The Dutch-Belgian (HOVON) study is slightly different,
since in one arm they use intermediate but nonmyeloablative
INTRODUCTION
doses of melphalan (two pulses of 70 mg/m2) without stem
cell support, and in the other arm they use the same regimen
This symposium covered three major topics: the comparison
followed by myeloablative treatment with cyclophosphamide
between single and double transplants, evaluation of residual
(12 mg/kg) and TBI. Although 453 patients have been
disease, and allogeneic transplantation including analysis of
enrolled into the study, only 255 have so far been random-
the role of donor-lymphocyte infusions (DLI) and nonmye-
ized after the initial four courses of VAD. The preliminary
loablative (mini-allogeneic) transplants.
results show that there were no significant differences
between the two arms for response rate, OS, or EFS.
Dr. Harousseau discussed best-option conditioning regimen.
Based on his data, high-dose melphalan alone is superior to
It could be concluded that, although so far the benefit of
the combination of melphalan and total body irradiation
double transplant is not clear and some of the studies pre-
(TBI) in terms of reduction in days of neutropenia and
sented require longer follow-up, at least two of the three
thrombocytopenia, transfusion, and days of hospitalization.
studies demonstrated a prolongation of disease control (EFS).
There is also a trend favoring melphalan alone in response
In addition, it should be mentioned that transplant-related
rate and survival. At present, the French group is exploring
mortality (TRM) was very low (<5%) in all three studies.
new conditioning regimens with higher doses of melphalan
or a combination with anti-interleukin-6.
The second topic was the evaluation of residual disease. Dr.
Robert Vescio, of Cedars-Sinai Comprehensive Cancer
The comparison of one versus two autologous transplants was
Center discussed why we need more sensitive techniques for
addressed in three presentations from French, Italian and
following tumor burden. He concentrated on the value of
Dutch groups. Dr. M. Attal analyzed the results of the Inter
molecular measurements of residual disease, using the pattern
Groupe Francais du Myéloma, which included 400 patients
of immunoglobulin gene rearrangement as the patient-specif-
less than 60 years of age with double randomization. The first
ic tumor marker and a highly sensitive polymerase chain
randomization compared one transplant conditioned with
reaction technique. He has observed that tumor burden
melphalan (140 mg/m2) plus TBI (8 Gy) with two trans-
rarely fell more than 2 logs after transplantation and that in
plants, one conditioned with melphalan only (140 mg/m2)
most patients' residual disease persists at the molecular level.
and the other identical to the first group. The second ran-
Dr. Vescio reported that changes from baseline (pretrans-
domization compared the use of bone marrow versus periph-
plant) in circulating tumor burden correlated with disease
eral blood (PB) stem cells. The results showed that double
relapse; he concluded that molecular monitoring may help
transplant supported with PB stem cells improves response
evaluate maintenance therapy programs, which will be need-
rate, event-free survival (EFS), and overall survival (OS).
ed to further improve treatment outcome in multiple myelo-
The achievement of complete response (CR) or very good
ma (MM).
partial response was the most important prognostic factor.
Regarding the current strategies of the French group, they
Dr. Jesus San Miguel discussed the role of multiparametric
have decided to stratify patients at diagnosis based on the
flow cytometry immunophenotyping for evaluation of
level of B2-microglobulin and cytogenetics (deletion of chro-
changes in plasma cell (PC) compartments following treat-
mosome 13). For low-risk patients, they will use a double
ment. This technique allows discrimination between normal
transplant and then randomly compare thalidomide and
and malignant PCs. Compared with chemotherapy, autolo-
pamidronate as maintenance therapy. For high-risk patients,
gous transplantation produced a significantly higher reduc-
they will intensify the dose of melphalan (220 mg/m2), or, if
tion in the number of myelomatous PCs together with a
a sibling donor is available, the second transplant will be
18

---

higher recovery of normal PCs. Moreover, while in MM
patients at diagnosis normal PCs are almost absent (<5%),
after treatment some patients showed more than 30% of
total PCs phenotypically normal and had longer progression-
free survival. Accordingly, it can be concluded that this new
method allows a sensitive evaluation of the PC compartment
and that, in MM patients, the recovery of high percentages
of normal PCs (a pattern similar to that observed in patients
with monoclonal gammopathy of undetermined significance)
is associated with a favorable outcome.
Three other presentations discussed the role of allogeneic
transplantation. First, Dr. Gösta Gahrton analysed experi-
ence reported by the European Bone Marrow Transplant
Group. The data indicate a marked improvement over the
years both in the reduction of TRM (from 46% to 30%) and
in OS (from 32% to 50% at 4 years). Another relevant
change was the shift from bone marrow to PB stem cells.
Although the latter has a clear benefit in terms of more rapid
engraftment, it is not so clear whether the survival benefit is
any better when PB is used. Dr. Gahrton discussed 54
patients transplanted with non-myeloablative regimens.
Although follow-up is too short, the potential efficacy of this
strategy, which is associated with reduced toxicity and TRM,
is evident. Recently, a Phase II/III study including autologous
transplantation followed by mini-allogeneic transplantation
has begun.
Dr. Lokhorst from Utrecht University addressed the graft-
versus-myeloma (GVM) effect induced by DLI in 39 MM
patients relapsing after allogeneic transplantation. The over-
all response rate was 52%, including 22% CR. Based on
these data, he concluded that the GVM effect has curative
potential. In the new HOVON trial, patients who undergo
allogeneic stem cell transplantation are randomized between
pre-emptive DLI 6 months after transplant and DLI adminis-
tered in the event of relapse.
Finally, Dr. William Bensinger of the Fred Hutchinson
Cancer Center reported on the Seattle experience with non-
ablative allogeneic transplants. The conditioning regimen is
based on their canine model and uses a very low dose of TBI
(200c Gy) together with fludarabine. This drug was intro-
duced because they observed a high rejection rate in patients
conditioned with TBI alone. At present they are using a
"tandem" transplant approach in which patients initially
undergo autologous transplant (to provide cytoreduction
with low morbidity) and then receive a mini-allogeneic
transplant, 2 to 4 months after the autotologous transplant,
to exploit the GVM effect. So far, 32 patients have received
transplants: 53% achieved CR and 81% are alive, with TRM
of only 19%. Although the results seem encouraging, the
incidence of chronic graft-versus-host disease is of concern,
and longer follow-up is needed to define the long-term out-
come for patients receiving non-myeloablative allogeneic
transplants. v
19

---

THALIDOMIDE: CLINICAL OUTCOMES
Brian G. M. Durie, M.D.
Cedars-Sinai Comprehensive Cancer Center, Los Angeles, California
mobilization/harvesting because of impaired collection of
peripheral blood stem cells. Higher likelihood of deep venous
thrombosis and blood test evidence of low thyroid function
occurred with thalidomide treatment. Results of this trial, as
well as of the ongoing IMiD 501 trial, are now awaited with
interest.
Five other investigators discussed the role of thalidomide,
both alone and in combination. Dr. Brian Durie from
Cedars-Sinai Cancer Center focused on the use of low-dose
thalidomide, ie, dosages from 50 mg (one capsule) to 400 mg
daily. This study, started in 1998, provided long-term follow-
INTRODUCTION
up and reflected significant clinical benefit with dosages of
50 to 200 mg daily. Three (33%) of the responders required
Dr. Kenneth Anderson of the Dana-Farber Cancer Center,
only 50 mg/day. Overall, 78% of the responses occur red with
Boston, who chaired the session, gave a brief overview of
200 mg/day or less of thalidomide. Although fewer patients
potential biologic effects of thalidomide and the new ana-
in this series (25% versus 75%) had received prior stem cell
logues such as the immunomodulatory drug IMiD 501. IMiD
transplants, response rates and survival were remarkably simi-
501 is currently being evaluated in a Phase I trial at Dana-
lar to those at Little Rock: 44% response rate overall and
Farber and in Little Rock. Thus far, IMiD 501 has been gen-
25% with at least 50% regression; 64% of patients were alive
erally well tolerated without evidence of neuropathy. The
at 2 years, with 22% still in remission. A major difference
appropriate dosage for Phase II trials is not yet established.
was the much lower toxicity with the low-dose schedule.
Nonetheless, ongoing peripheral neuropathy was a significant
HIGHLIGHTS
concern, especially with therapy for more than 1 year.
Thalidomide combined with pulse dexamethasone (40 mg
The keynote speaker was Dr. Bart Barlogie of the Arkansas
daily for 4 days twice per month), with or without added
Cancer Research Center. Results with "total therapy"
Biaxin (clarithromycin 500 mg twice daily), was used to treat
(including double stem cell transplantation) indicate a 50%
failing or relapsing patients. Thalidomide plus dexametha-
complete remission rate in patients with very good prognosis;
sone produced responses in three of seven patients (43%),
such patients can live for 10 years or more. Conversely,
with less than a 50% response with thalidomide alone. The
patients with chromosome 13 deletion, even with total ther-
three-drug combination BLT-D (Biaxin, low-dose thalido-
apy, had a very poor prognosis. A focal point of the presenta-
mide, dexamethasone) produced benefits in 4 of 10 patients
tion was the role of thalidomide, first as an agent for relapse
(40%) who were failing with the two-drug combination, con-
therapy and then incorporated into the new "Total Therapy
firming added benefit with Biaxin in this setting. Further
II" protocol. Follow-up of 169 patients with relapsing disease
studies with low doses of thalidomide alone and in combina-
treated with thalidomide as a single agent showed evidence
tion were recommended.
of response in one third of patients treated with a q2wk dose-
escalation schedule with 200 mg/day as the starting dosage.
With these encouraging results in relapsing patients, there
Toxicity was significant with dosages of at least 400 mg/day.
was considerable interest in the efficacy of thalidomide as
Overall survival was 60% at 2 years from the start of thalido-
part of primary therapy for previously untreated disease. Data
mide therapy, with 20% of patients still in remission at 2
from M.D. Anderson Cancer Center were presented by Dr.
years. Patients with good prognostic factors did the best,
Donna Weber and from the Mayo Clinic by Dr. Vincent
including low serum b2 microglobulin, LI% (labeling index)
Rajkumar. Both groups studied thalidomide plus dexametha-
and CRP (C-reactive protein) as well as no major chromo-
sone. The M.D. Anderson study evaluated thalidomide 400
some abnormalities.
mg combined with dexamethasone using intensive 4-day-
on/4-day-off pulses. Eleven of 16 patients (69%) with symp-
Based on these data, thalidomide was incorporated into
tomatic (active), previously untreated myeloma responded.
"Total Therapy II" at 400 mg daily. Results of this trial are
Follow-up is still short. Patients with "asymptomatic disease"
not yet available. Anticipated accrual is 600 patients.
(a subgroup of patients with early disease, identified by the
Thalidomide is now stopped during stem cell
MD Anderson team) were also treated with thalidomide
alone; 10 of 28 (36%) had an initial response duration of
20

---

more than 1 year. Toxicity in both groups was significant,
mg once weekly.
with neurologic toxicity occurring in approximately 60%
including unsteadiness/lightheadedness, tremors, psychosis,
Enteric-coated aspirin (81 mg/day) was added as a preventive
and peripheral neuropathy. The rapid responses in these
for clotting complications. The 55 patients were a mixed
studies indicated a direct cytoreductive effect of thalidomide
population in terms of both diagnosis (6 Waldenstrom's, 49
rather than an antiangiogenic mechanism. This aspect was
myeloma) and disease status (9 untreated, 46 previously
commented on by several speakers as well as in posters pre-
treated with a variety of regimens). A very high response
sented at the meeting.
rate was observed (80% to 90% depending on population).
The data indicate that BLT-D is superior to LT-D, but an
The Mayo Clinic study included 26 patients with active, pre-
ongoing randomized trial will be necessary to fully assess the
viously untreated myeloma. Twenty of them (77%) respond-
impact of the added Biaxin. It should be noted, however,
ed to thalidomide with a dose-escalating schedule (200-800
that evaluation of Biaxin, both in vitro in myeloma cultures
mg/day) combined with dexamethasone using the same
and added to therapy for failing patients as part of the
intensive 4-day-on/4-day-off protocol as used by the M.D.
thalidomide/dexamethasone trial at Cedars-Sinai Cancer
Anderson group. Toxicity was again significant and included
Center, supports the notion that Biaxin provides additional
life-threatening skin rashes in three patients. Following this,
benefit. Although this is in part because of synergy with
the thalidomide dosage was kept at 200 mg/day without dose
steroids, the definite mechanism of benefit awaits full expla-
escalation. Other toxicities included syncope, sedation, con-
nation.
stipation, arrhythmia, and myalgia. In the M.D. Anderson
group, patients with smoldering myeloma were treated with
Dr. Mohamed Hussein from the Cleveland Clinic reviewed
thalidomide alone (200­800 mg/day), and six patients (38%)
thalidomide toxicities and outlined plans for ongoing moni-
responded (c.f. 36% Houston). Although angiogenesis was
toring and management. Continuing thalidomide treatment
evaluated serially, the direct relationship to thalidomide
in the face of progressive peripheral neuropathy (especially
response was unclear.
numbness and difficulty with fine movements such as but-
toning clothing) is an important dilemma. How safe is it to
Perhaps the most consistent observation was the overall effi-
stop thalidomide? Clearly, some patients taking thalidomide
cacy of thalidomide. When it is used as monotherapy,
alone can stop the drug once remission is achieved and
responses occur in 25% to 40% of patients both at relapse
maintain remission for more than 6 months. However, oth-
and with previously untreated disease. As first pointed out
ers have early evidence of increasing myeloma protein levels.
in the low-dose study from Cedars-Sinai Cancer Center,
Further studies are required to assess stopping versus low-dose
patients with kappa light chain subtype disease are more
maintenance of some sort (eg, 50 mg 2 or 3 times per week).
likely to respond. The M.D. Anderson data and large studies
from Greece (Dimopoulos) and Italy (Boccadoro) show the
The greatest anticipation focuses on the early IMiD trials.
same trend.
What toxicities will there be? What level of benefit will be
achieved? How soon will broader Phase II trials begin?
Twenty abstracts in the poster session dealt with thalidomide
Thalidomide and its analogues represent a rich area for clini-
treatment. Results were presented from around the world,
cal research and major new treatment advances to improve
including Europe (United Kingdom, Italy, France, Germany,
outcomes for patients with myeloma and related disease. v
Poland) and Asia (India, Australia), as well as from the
United States. Efficacy and toxicities were remarkably simi-
lar between groups. Combination with steroids clearly
enhances short-term efficacy. Other combinations may also
have merit, including with high-dose Cytoxan and in DT-
PACE. Results with TAD were presented, in which thalido-
mide (T) replaces vincristine (V) in the VAD regimen. At
Memorial Sloan­Kettering Cancer Center, early results show
significant efficacy. Coumadin was added as an anticoagu-
lant to prevent deep venous thrombosis. Further follow-up is
required.
Perhaps the greatest interest concerned the BLT-D protocol
presented as a poster by Dr. Morton Coleman of Cornell.
Based on prior evidence of efficacy with Biaxin 500 mg PO
BID, (twice daily) Biaxin was added to low-dose thalidomide
(50 mg daily) plus dexamethasone at a starting dosage of 40
21

---

VACCINES STRATEGIES IN MULTIPLE MYELOMA
Håkan Mellstedt, M.D., Ph.D.
Karolinska Hospital/Karolinska Institute,Stockholm, Sweden
different peptide sequences of the complete V region can be
presented on MHC molecules, the whole idiotype should be
used as vaccine and not only CDRs.
The induction of idiotypic immunity is probably induced by
the secreted myeloma protein taken up by dendritic cells
(DCs), which present MHC class II- and I-restricted peptides
to the immune system and induce CD4 and CD8 responses.
The relative contribution of different T-cell subsets to the
eradication of myeloma cells is not clear. Both CD4 and
CD8 T-cells have been considered important to tumor rejec-
tion, and both subsets probably contribute. Dr. Freda
INTRODUCTION
Stevenson of the University of Southhampton in England
proposed that there might be a CD4 tolerance for the idio-
Myeloma cells express several tumor antigens that might be
type induced by the long-term presence of tumor cells.
candidates for use in vaccine development. At present, focus
However, in colon carcinoma with a high secretion of CEA,
is on idiotypic proteins. These proteins, coupled to carriers or
which might be analogous to myeloma, it had been claimed
loaded onto autologous dendritic cells, have been used for
that there is CD8 T-cell tolerance. These contradictory
vaccination together with adjuvant cytokines. Immune
results probably indicate that we do not have complete infor-
responses as well as clinical effects have been noted in early
mation and that there might be great variation, supporting
trials. A low tumor burden and a well-preserved immune sys-
the notion that a broad cellular immune response should be
tem may be prerequisites for success of the vaccination
the goal of vaccination.
approach. In the future, improved vaccine preparations and
protocols will facilitate the development of a therapeutic
There are other antigens besides the idiotype that can be used
vaccination strategy. Large clinical trials are needed to estab -
as vaccine candidates, eg, mucins. At present, however, the
lish the clinical efficacy of idiotype vaccination. Such trials,
focus is entirely on clone-specific idiotypic immunoglobulin.
however, should not be initiated until a promising vaccina-
tion protocol has been developed. With this in mind, it is
Induction of immunity in myeloma patients, which should
anticipated that by the end of this decade vaccination will
have the potential to eradicate tumor cells, is a great chal-
be an established therapeutic approach in myeloma.
lenge. Dr. Mellstedt noted that the goal is not only to have a
good antigen presented in a proper way but also to have a
HIGHLIGHTS
well-functioning immune system. There might be many hur-
dles to overcome in developing a vaccination strategy. Dr.
The tumor clone in multiple myeloma consists of monoclon-
Massaia of the University of Torino in Italy suggested that
al myeloma B cells (plasma cells and late B-lymphoid cells)
the antigen-presenting cells and DCs may be defective in
synthesizing the immunoglobulin of the B cell of origin. The
myeloma. Patients with myeloma, even at a low stage, who
idiotypic determinants of the immunoglobulin can be consid-
are chemotherapy naive have immune T-cell dysfunction
ered a well-defined tumor antigen. Peptides of the idiotypic
measured as a low capability to secret cytokines and a defect
region, complementarily-determining regions (CDRs) and
expression of T-cell­signaling molecules. This information is
framework regions, can be presented on MHC class II and I
important to the further clinical development of effective
molecules of myeloma cells and thus be recognized by both
vaccines, as measures have to be taken to correct defects in
CD4 and CD8 lymphocytes.
T-cell signaling. This might be achieved by simultaneous
administration of cytokines and high doses of vitamin E,
Antigens that may induce spontaneous immunity might be
which have also been suggested to correct T-cell dysfunction.
used preferentially as vaccine candidates. The idiotypic
immunoglobulin region of myeloma cells can induce natural
Collectively, these prevaccination results indicate that the
T-cell immunity, including cytotoxic T lymphocytes that
idiotype is an appropriate antigen, but it is important to
might lyse the autologous tumor cells. This indicates that the
select patients with well-preserved immune systems to be
idiotype might be an immunodominant epitope and a good
able to induce adequate immune responses.
candidate for use in vaccine development. Furthermore, as
22

---

The idiotype can be delivered as a protein antigen, as protein
much more sensitive technique for cytokine assay is real-time
loaded onto isolated autologous dendritic cells, or as DNA
polymerase chain reaction (PCR), which detects fewer
presented in a plasmid. There is debate regarding which is
cytokine-specific T-cells and requires fewer cells. Moreover,
the best vaccine preparation, protein or plasmid DNA.
several cytokines can be assayed easily with real-time PCR,
Further clinical development will reveal which antigen
which gives a better prediction of the functional capability of
preparation is the most powerful in inducing clinically effec-
idiotype-specific T-cells. A clinically sufficient T-cell
tive immunity. When one uses the idiotypic protein as an
response may not consist of more than one or two specific T-
antigen, it is clear that granulocyte-macrophage colony-stim-
cells per 10,000, which cannot be detected by ELISPOT or
ulating factor (GM-CSF) is necessary; otherwise, CD4 and
flow cytometry. Therefore, it is recommended that prolifera-
CD8 T-cell response will not be induced. In a preclinical
tion assay, ELISPOT, and real-time-PCR be used as readout
model described by Dr. Stevenson, plasmid-expressing idio-
systems to have a good chance of adequately monitoring an
type DNA alone (producing idiotypic protein in vivo) did
idiotype-specific immune response. In addition, patients'
not have a clinical effect. Fusion to fragment C of tetanus
immune T-cell dysfunction should be evaluated and correlat-
toxid was necessary; this construct induced protective immu-
ed to the capability of the patient to mount an idiotype-spe-
nity; CD4 cells were important. Plasmid-idiotype DNA and
cific immune response. These results might yield more infor-
GM-CSF were not tested in this model. Idiotype fused to
mation regarding why patients may or may not mount idio-
fragment C of tetanus toxid was necessary, but GM-CSF
type-specific immune responses and form a basis for selection
might have a similar effect. Fusion of fragment C of tetanus
of patients for idiotype vaccination protocols.
toxid to CEA was necessary to induce an anti-CEA response,
whereas plasmid CEA alone had no effect. Dr. Mellstedt
Numerous studies were presented using idiotype loaded onto
reported that vaccination with protein CEA alone induced
DCs together with GM-CSF. On average, 31% (CI95%: 19%-
no or only weak immunity, but protein CEA in combination
43%) mounted idiotype-specific proliferative responses. The
with GM-CSF induced a strong humoral (IgG) response and
patients were in advanced stage or in remission after
CD4 as well as CD8 T-cell responses. It has also been shown
chemotherapy. The induction of a proliferative response sug -
that GM-CSF facilitated the MHC class I antigen-presenta-
gested a CD4 T-cell response that did not support an assump-
tion pathway and, thereby, the induction of cytotoxic T lym-
tion of the presence of tolerant CD4 T-cells. Interesting pr e-
phocytes, which may be important in rejection of tumor
liminary clinical observations were also made. Patients who
cells. An increasing number of reports from different experi-
mounted immune responses had a longer time to disease pro-
mental systems indicate that CD4 cells are extremely impor-
gression than patients without immune responses. Those
tant for tumor rejection. The idiotypic protein isolated from
with preexisting idiotype responses had the longest time to
serum should, from a practical point of view, facilitate the
disease progression. The last observation is in line with one
use of the idiotype as a vaccine preparation.
by Dr. Mellstedt that patients with preexisting immune
responses developed idiotype-specific responses more rapidly
In most cases where ex vivo DCs have been used for loading
and that the magnitude was higher than in patients with no
the idiotypic protein, monocyte-derived DCs were selected.
preexisting immune response. Dr. MacKenzie also reported
However, with a new technological platform, blood DCs,
that patients with minimal tumor burden after chemotherapy
which are a more efficient immunogen than monocyte DCs,
had a longer time to disease progression than those with
can be isolated. Dr. Hart stated that sufficient numbers of
"bulky" tumor, and the shortest time was noticed for patients
blood DCs can be isolated from one leucapheresis product to
who had relapsed after transplant. These data indicate that
produce 30 doses of vaccines, which can be cryopreserved.
immune T-cell dysfunction had an impact on the clinical
This new purification platform may facilitate the use of DC-
efficacy of idiotype vaccination. Dr. Lacy observed five clini-
based vaccines.
cal responses (three complete remission, one new complete
remission, and one partial remission) and five patients with
Monitoring of an idiotype-specific immune response is not
stable disease for a long time out of 17 patients with residual
yet standardized. It is important that all research groups
disease enrolled in a vaccination trial after transplantation.
involved in vaccine development use the same technologies
with standardized protocols. Proliferation assay (3H-thumi-
Dr. Nikhil Munshi of the University of Arkansas presented
dine incorporation) is used by all groups. However, to facili-
interesting data: He used monocyte-derived DCs loaded with
tate picking up idiotype-specific T-cells, several assays should
the idiotypic protein and given intravenously. Only a few
be applied; these T-cells might be in different phases of the
patients developed immune responses, and there was no clin-
cell cycle as well as in maturation stages and of different sub-
ical response. Those who developed immune responses had
sets that may not be detected by proliferation assay only,
received the highest dose of DCs. In a subsequent study by
which mainly detects CD4 T-cells. Cytokine assays should be
the same group, patients received the highest dose of DCs (1
added. ELISPOT estimates the frequency of cytokine-secret-
ing cells, and both type I and II cytokines can be analyzed. A
Please see page 25
23

---

IMMUNOTHERAPIES AND CONTROL OF RESIDUAL DISEASE
Brian G.M. Durie, M.D.
Cedars-Sinai Comprehensive Cancer Center, Los Angeles, California
cells being used for stem cell transplantation. He focused on
the use of SLT-1 (shiga-like­ toxin), a bacterial toxin pro-
duced by certain strains of Eschericia coli that can infect the
gastrointestinal tract. SLT-1 can destroy myeloma cells in
vitro while sparing normal bone marrow stem cells. Testing
of SLT-1 in transplant patients is just beginning.
The final presentation was by Dr. Mitterer from Italy. He and
his colleague, Dr. Straka of Ludwig Maximillians University
in Germany, used monoclonal antibodies against B lympho-
cytes (presumed myeloma precursor cells) to clear stem cell
preparations of residual contaminating myeloma precursor
INTRODUCTION
cells. Effects in 55 patients were compared with those in 57
patients in whom no purging was performed. Preliminary
Dr. Douglas Joshua, of the Royal Prince Albert Hospital in
data indicate that both length of remission and overall sur-
Australia, chaired this session. Four speakers presented, two
vival are improved with use of this technique. Further fol-
dealing with the new immune therapy target antigens
low-up is required, and additional studies are planned. It
hTERT and TRAIL and two with new methods to purge
should be noted that overall, purging still needs to be estab-
stem cells as a part of high-dose chemotherapy with stem cell
lished as a useful technology. Results thus far from different
transplantation.
international groups have been conflicting. Although purg-
ing definitely yields a "cleaner" stem cell product, it is not so
HIGHLIGHTS
clear to what extent remission and survival are impacted. v
Dr. Joachim Schultze of the Dana Farber Cancer Institute
discussed the significance of the hTERT antigen, the catalyt-
ic subunit of telemerase and enzyme that is present on the
surface of 85% of cancer cells including myeloma. hTERT
confers "immortality" to growing malignant cells. The aim is
to trigger immune T lymphocytes from myeloma patients to
react selectively against this surface hTERT protein and
thereby inhibit the immortality potential of actively growing
myeloma cells. The method is to develop a panel of suitable
surface antigens that will function as a sufficiently strong
trigger to produce an effective immune response. This work
is in its early stages.
The second presentation was by Dr. Steve Treon, also from
Dana Farber, who discussed his recent studies with TRAIL
(TNF-related apoptosis-inducing ligand). Myeloma cells
have surface receptors for TRAIL. Administration of syn-
thetic TRAIL (produced by recombinant genetic engineer-
ing) causes myeloma cells to undergo cell death, or apoptosis.
Other agents that affect signal-transduction pathways, such
as the new proteasome inhibitor PS-341, an NFkB inhibitor,
enhance this induction of cell death. Again, these studies are
in the early stages, and patient trials (with TRAIL) will be
awaited with interest.
Dr. Andrew Belch of the University of Alberta in Canada
presented results of studies on novel strategies to purge stem
24

---

Vaccines Strategies in Multiple Myeloma -
are usually low-avidity antigens, and it is necessary to do
Continued
repeated immunization when using them.
x 107) used in the previous study, but DCs were given subcu-
Dr. Kwak and colleagues showed that sibling donors to recip-
taneously. Dr. Qing Yi, also of the University of Arkansas,
ients of allogeneic transplants, immunized with the patient's
reported that one patient out of five entered OR (objective
idiotype, developed idiotype-specific CD4 cells, which could
remission) (>50% reduction of the M component), and the
be transferred during the transplant to the recipient. This
remaining four had stable disease. This indicates the impor-
mode of idiotype immunization should circumvent CD4 tol-
tance of the dose and route of administration of DCs.
erance. Studies are in progress by Drs. Kwak and Stevenson.
However, this approach is only available for a restricted
Dr. Massaia and colleagues had vaccinated 15 patients in
number of myeloma patients.
remission after chemotherapy using idiotype conjugated with
KLH in combination with GM-CSF. Eighty-five percent
We are at the beginning of development of a therapeutic
mounted idiotype-specific delayed-type hypersensitivity reac -
vaccine strategy in myeloma. At present, focus is on the idio-
tion (reflecting a CD4 T-cell response). No proliferative
type as target structure, but other antigens are also available.
response was recorded. The reason for this discrepancy is not
Induction of idiotype-specific CD4 and CD8 T-cells is neces-
clear, but it favors the notion that CD4 T-cells may not be
sary. The idiotypic protein coupled to KLH or alum in conju-
tolerated by myeloma patients. These patients had a median
gation with GM-CSF may induce idiotype-specific CD4 and
time to relapse after start of vaccination of 35 months versus
CD8 T-cells. Idiotype loaded onto dendritic cells also induce
24 months for a matched population. The difference was not
an immune response and clinical effects. Data regarding the
significant. These patients also had abnormal T-cell reper-
clinical efficacy of DNA vaccines in humans will come in
toires, suggesting abnormal T-cell function and indicating
the near future. It is very important that research groups test
that the population was not optimal for vaccination. Drs.
various concepts with regard to vaccine preparations, adju-
Munshi and Kwak also showed that the idiotypic protein
vant cytokines, and scheduling. As soon as we have opti-
conjugated with KLH and given together with GM-CSF
mized the vaccination procedure, extended clinical trials are
induced an idiotype-specific T-cell response (CD4/CD8) in
urgently needed, but it is important to select optimal
about 75% of the patients, and those patients had signifi-
patients, who should have a minimal disease burden and
cantly longer relapse-free survival than patients who did not
well-preserved immune systems.
mount idiotype-specific T-cell responses.
Another adjuvant cytokine of importance in vaccine develop-
Since the first results on idiotype immunization were reported
ment is interleukin (IL)-12. This cytokine leads to a type I T-
in 1994,a great deal of progress has been made. The progress
cell response and amplifies the magnitude of the immune
may seem slow, but within this decade, we should have a thera-
response. In studies performed by Dr. Mellstedt and colleagues,
peutic vaccine concept in clinical use in multiple myeloma. v
about 90% of the patients mounted idiotype-specific immune
responses when the idiotypic protein (absorbed onto alum)
was given together with GM-CSF and IL-12; 45% mounted
immune responses when IL-12 was given alone and 70% when
GM-CSF was given as the only adjuvant cytokine. The high-
est amplitude of immune response was seen in patients receiv-
ing the combination of IL-12 and GM-CSF. Only one major
response (>50% reduction of the M component) was seen in
this population of vaccinated stage I myeloma patients.
However, in 50% of the studied patients, circulating tumor
cells, measured by real-time PCR and VH-CDRIII specific
primers and probes, gradually disappeared. The kinetics of the
tumor response speak against a redistribution phenomenon of
tumor cells in blood circulation. The result indicates that the
tumor-specific immune response may not be able to reduce a
large tumor burden but may affect minimal disease. The study
also showed that repeated immunizations were necessary, as it
may take up to 8 months (eight immunizations) before an
idiotype-specific immune response can be evoked. Moreover,
preliminary data indicate that there seems to be no tolerance
induced by repeated immunizations. This is not to be expected
when one uses a self-antigen as an immunogen. Self-antigens
25

---

NEW THERAPIES
Kenneth C. Anderson, M.D.
Dana-Farber Cancer Center, Boston, Massachusetts
Rotterdam) further discussed mechanisms whereby MM cells
escape from chemotherapy. His group was among the first to
show that MM cells can become educated, or drug resistant,
via the induction of pumps inside tumor cells that exclude
chemotherapy and thereby confer protection. He described
lung resistance protein (LRP), which is present in MM cells
even before treatment. LRP protects tumor cells against
melphalan, an agent commonly used to treat MM. LRP is
also called major vault protein (MVP); together with other
proteins, it appears to block transport of drug from the tumor
cell surface to the cytoplasm inside, eventually inhibiting
their entry to the nuclear compartment. Understanding how
INTRODUCTION
MVP works to protect tumor cells against chemotherapy
therefore offers possible new methods to interrupt its effects.
Novel biologically based therapies target both the multiple
myeloma (MM) cell and its microenvironment.
Dr. Sergio Giralt and colleagues have tested a novel radionu-
Understanding the cellular and molecular mechanisms of
cleide called homium 166Ho-DOTMP, which homes to the
their anti-MM activity has already and will continue to aid
bone and delivers relatively higher doses of radiation to the
in the identification of novel targets and strategies to over-
bone marrow site of MM than to normal organs. He
come drug resistance and improve patient outcome.
described a study evaluating the safety and efficacy of either
166Ho-DOTMP plus high-dose melphalan or of 166Ho-
HIGHLIGHTS
DOTMP plus high-dose melphalan plus total body irradia-
tion, followed by autologous peripheral blood stem cell trans-
Dr. William S. Dalton and coworkers at the H. Lee Moffitt
plantation. High response rates were seen, which need to be
Cancer Center have studied several mechanisms whereby
further confirmed in ongoing clinical trials. Continuous
MM cells themselves resist chemotherapy, including reducing
bladder irrigation appeared to be necessary to avoid side
uptake of drug into the tumor cell, altering the breakdown of
effects, including bloody bladder inflammation or kidney-
the drug once it is in the patient, altering the target of the
related complications, the hemolytic uremic syndrome.
drug within the MM cell, and repairing the drug-induced ill
effects in MM cells. Dr. Dalton discussed two factors within
Dr. Ozaki from the University of Tokushima (Japan)
the bone marrow microenvironment of MM cells that also
described a protein called HM-1.24 on the surface of MM
allow them to resist chemotherapy. First, cytokines such as
cells that can serve as a target for their selective removal.
interleukin-6 (IL-6) allow MM cells to resist death by turn-
He showed data on an antibody directed at HM-1.24 that
ing on the JAK/STAT circuit and ultimately increasing the
was humanized and so could theoretically work much like
protective Bclx protein in MM cells. In contrast, blocking
Rituxan serotherapy, which targets B-cell lymphoma. This
this circuit using the drug AG490 can restore sensitivity to
antibody or serotherapy approach has great potential, as once
drug-induced MM cell death. However, in some cases,
MM cells have reacted with HM1.24 antibody, they are
AG490 can induce dormancy of tumor cells and actually
marked for removal by the patient's own immune system.
then protect them from death, demonstrating the need to
This treatment approach warrants further study to extend
study the MM cell in its natural bone marrow environment.
these promising preliminary findings.
Importantly, direct contact and binding of the MM cell to
bone marrow cells and noncellular proteins in the bone mar-
Dr. John Lust and coworkers from the Mayo Clinic presented
row results in cell adhesion-mediated drug resistance.
studies on chemoprevention, the use of drugs to prevent the
Delineating those circuits mediating this protective effect
evolution of MM from the precursor lesion called monoclon-
may suggest treatments that are based on blocking adhesion
al gammopathy of undetermined significance (MGUS).
of MM cells to bone marrow cells and proteins as well as
Their working hypothesis is that MM patients' bone marrow
interrupting those circuits triggered by adherence that
produces IL-1, which in turn stimulates IL-6 production.
enhance MM cell survival.
IL-6 is the major growth and survival factor for human MM
cells, and its secretion correlates with progressively active
Dr. Pieter Sonneveld and colleagues (University Hospital,
MM; therefore, blocking this process might inhibit develop-
26

---

ment of active MM. He presented the design of ongoing tri-
Fourth, 2-methoxy estradiol (2ME) is a hormone that
als using two drugs, DHEA to downregulate IL-6 and Biaxin
induces death of drug-resistant MM cells and also inhibits
to downregulate IL-1. These studies may provide the
angiogenesis in the bone marrow. This drug is under testing
framework for the use of inhibitors of IL-1 or IL-6 to block
at the Mayo Clinic and at Dana-Farber Cancer Institute.
progression of MM or, importantly, to prevent the develop-
Arsenic trioxide also induces death of drug-resistant MM
ment of MM in those patients with MGUS destined to
cells and acts to inhibit new blood vessel and cytokine pro-
progress to MM.
duction in the bone marrow; early clinical testing in MM
patients is now ongoing. Tumor necrosis factor alpha (TNF
Finally, Dr. Kenneth Anderson presented data on several
) is another novel therapeutic possibility in MM, since it
classes of novel drugs that have in common targeting not
increases the binding affinity of MM cells to bone marrow
only the tumor cell but also the microenvironment. These
cells and related protection against drugs resulting from this
drugs directly induce MM cell death or dormancy and also
binding. TRAIL/Apo2L is a drug that also induces death of
inhibit the ability of MM cells to localize in bone marrow,
drug-resistant MM cells without adversely affecting normal
the production of cytokines in the bone marrow that pro-
blood cells; it will soon enter clinical testing in MM and
mote MM growth and survival, and new blood vessel forma-
other cancers.
tion or angiogenesis and, in some cases, stimulate the
patient's own immune responses against MM.
Finally, AE-491 is a derivative of shark cartilage that is a
very potent inhibitor of angiogenesis and tumor growth; a
First, thalidomide and its potent immunomodulatory drug
multicenter international trial of this agent in MM has
(IMiD) derivatives directly induce death or dormancy, even
already begun. These drugs are examples of biologically
in drug-resistant MM cells and patient cells; abrogate
based therapies with novel mechanisms of action against the
increased IL-6 and vascular endothelial growth factor
MM cell and/or the bone marrow microenvironment that are
(VEGF) secretion triggered by MM cell binding to bone
or will soon be in clinical trials. They represent bench-to-
marrow cells; stimulate autologous natural killer cell-mediat-
bedside research in a new treatment paradigm for MM tar-
ed anti-MM immunity; and inhibit new blood vessel forma-
geting both the tumor cell and its bone marrow milieu and
tion in MM bone marrow. They add to dexamethasone,
offer great promise to improve outcomes for patients with
both in the lab and when used together to treat patients. In
MM. v
a Phase I dose-escalation trial designed to define the highest
tolerated dose of IMiD, 7 of 11 patients whose disease was
unresponsive to any other therapy have achieved either sta-
bilization or response. Importantly, side effects have so far
been minimal.
Second, NF-B activation is a switch that mediates growth,
survival, and drug resistance in MM cells, as well as the
increase in IL-6 production and secretion triggered by adher-
ence of MM cells to bone marrow cells. Proteasome
inhibitor PS-341 blocks this switch and in the laboratory
kills even MM cells resistant to all known therapies. These
drugs block the production of IL-6 and other cytokines in
the bone marrow that the MM cell uses to grow and survive.
Importantly, PS-341 blocks adherence of MM cells to bone
marrow cells, which makes them susceptible to killing by
chemotherapy. Excitingly, early Phase II trial results suggest
that patients with MM relapsing after conventional treat-
ments can benefit from proteasome inhibitor therapy, and
ongoing trials will rapidly define their ultimate utility.
Third, MM cells secrete VEGF, which may account, at least in
part, for the increased bone marrow angiogenesis in MM; it
also triggers growth and motility of MM cells in the bone mar-
row. Based on both the direct effect of VEGF on MM cells
and its effects on the bone marrow neighborhood, inhibitors of
VEGF are soon to be tested clinically against MM.
27

---

INTERNATIONAL MYELOMA FOUNDATION
PROGRAMS AND SERVICES
"One person can make a difference,
includes The IMF Patient Handbook and Prof.
Two can make a miracle."
Brian G.M. Durie's Concise Review of the Disease
Brian D. Novis
and Treatment Options as well as information on
IMF Founder
other IMF publications and services.
Multiple myeloma is a little-known, complex and
MYELOMA TODAY
often misdiagnosed bone marrow cancer that
Stay in the loop with Myeloma Today, the IMF
attacks and destroys bone. Myeloma affects approx-
newsletter published six times per year. Myeloma
imately 75,000-100,000 people in the United
Today covers a broad range of topics, offering
States, with more than 14,500 new cases diagnosed
up-to-date information of use to both patients
each year. While there is presently no known cure
and physicians.
for myeloma, doctors have many approaches to help
myeloma patients live better and longer.
SUPPORT GROUPS
The IMF works with a network of more than 50
The IMF was founded in October 1990 by Brian
multiple myeloma support groups from around the
and Susie Novis shortly after Brian's myeloma
world. To find the support group nearest you, con-
diagnosis at the age of 33. It was Brian's dream
tact the IMF.
that future patients would have easy access to
medical information and emotional support
ONLINE MYELOMA FORUM
throughout their battle with myeloma. He estab-
The IMF Internet Discussion Group hosts an
lished the IMF with the three goals of Treatment,
online support group where users can log on and
Education and Research. He sought to provide a
share thoughts and experiences. Visit www.myelo-
broad spectrum of services for patients, their fami-
ma.org/listserve.html to access this forum.
lies, friends and healthcare providers. Although
Brian died four years after his initial diagnosis, his
PATIENT-TO-PATIENT DIRECTORY
dream didn't. Today the IMF reaches out to an
The IMF maintains a confidential worldwide
international membership of more than 78,000.
directory of patients and family members that can
The IMF was the first organization dedicated solely
help put you in touch with IMFers in your area.
to the field of multiple myeloma, and today
PATIENT & FAMILY SEMINARS
remains the largest.
Each year the IMF holds educational Patient &
The IMF provides programs and services to aid in
Family Seminars in cities around the world. These
the research, diagnosis, treatment and management
meetings enable patients and their families to
of myeloma. The IMF ensures that no one must
learn about the latest developments in myeloma
brave the myeloma battle alone. We care for
from a panel of world-renowned myeloma experts.
patients today, while working toward tomorrow's
The IMF Patient & Family Seminars are also a
cure.
great opportunity to meet others touched by
myeloma.
HOW CAN THE IMF HELP YOU?
RESEARCH
TOLL-FREE HOTLINE
The IMF research grant program funds both junior
The IMF Hotline (800) 452-CURE, toll-free
and senior investigators. Created in memory of
throughout the US and Canada, provides immedi-
IMF founder Brian D. Novis, the grants support
ate support and information.
cutting edge and novel research into better treat-
ment, management, prevention and ultimately, a
INTERNET ACCESS
cure for myeloma.
At www.myeloma.org, you'll find 24-hour access to
in-depth information about multiple myeloma as
PHYSICIAN SERVICES
well as information about the IMF and our educa-
The IMF organizes Clinical Conferences, Expert
tion and support programs.
Research Roundtables and Scientific Workshops to
keep physicians and researchers abreast of
INFORMATION PACKAGE
advances in the field of myeloma. Several IMF
The IMF Info Pack provides comprehensive infor-
programs offer Continuing Medical Education
mation on treatment options and disease manage-
(CME) credit.
ment. Available free of charge, the Info Pack
28

---

International Myeloma Foundation
The IMF is dedicated to improving the quality of life of
myeloma patients while working toward prevention and a cure.
UNITED STATES OF AMERICA
12650 Riverside Drive, Suite 206
North Hollywood, CA 91607-3421
U.S.A.
(800) 452-CURE
(818) 487-7455
(818) 487-7454 Fax
E-Mail: TheIMF@myeloma.org
Website: www.myeloma.org
UNITED KINGDOM
9 Gayfield Square
Edinburgh EH1-3NT
U.K.
0131-557-3332
0131-556-9720 Fax
E-Mail: TheIMF@myeloma.org.uk
Website: www.myeloma.org.uk
Sponsorship provided with unrestricted educational grants by Celgene Corporation and NeoRx Corporation.
© 2001 International Myeloma Foundation

---