CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS

Chromosome 13 abnormalities identified by FISH analysis and serum

␤2-microglobulin produce a powerful myeloma staging system
for patients receiving high-dose therapy
Thierry Facon, Hervé Avet-Loiseau, Gaëlle Guillerm, Philippe Moreau, Franck Geneviève, Marc Zandecki, Jean-Luc Laı̈,
Xavier Leleu, Jean-Pierre Jouet, Francis Bauters, Jean-Luc Harousseau, Régis Bataille, and Jean-Yves Mary
on behalf of the Intergroupe Francophone du Myélome (IFM)

A careful prognostic evaluation of pa-
tients referred for high-dose therapy (HDT)
is warranted to identify those who maxi-
mally benefit from HDT as well as those
who clearly fail current HDT and are can-
didates for more innovative treatments. In
a series of 110 patients with myeloma
who received HDT as first-line therapy,
times to event (disease progression and
death) were studied through proportional
hazard models, in relation to different
prognostic factors, including a chromo-
some 13 fluorescence in situ hybridiza-
tion (FISH) analysis using a D13S319
probe. ⌬13 was detected in 42 patients
(38%). Follow-up time among surviving
patients and survival time were 48 ⴞ 3
and 51 ⴞ 7 months, respectively (me-
dian ⴞ SE). In the univariate analysis, ⌬13
was the most powerful adverse prognos-
tic factor for all times to event, especially
for the survival time (P < .0001) and was
followed by ␤2-microglobulin (␤2m) levels
2.5 mg/L or higher (P ⴝ .0001). The com-
parison of survival prognostic models
including ␤2m 2.5 mg/L or greater and
another factor favored the ⌬13/␤2m com-
bination. In 22 patients (20%) with no
unfavorable factor, the median survival
time was not reached at 111 months. In
contrast, among 55 patients (50%) with
one unfavorable factor and 33 patients
(30%) with 2 unfavorable factors, median
survival times were 47.3 ⴞ 4.6 months
and 25.3 ⴞ 3.2 months, respectively
(P < .0001). We conclude that ⌬13, ad-
equately detected by FISH analysis, is a
very strong factor related to poor sur-
vival, especially when associated with a
␤2m level of 2.5 mg/L or higher. Routine
FISH ⌬13 assessment is strongly recom-
mended for patients considered for
HDT. (Blood. 2001;97:1566-1571)
© 2001 by The American Society of Hematology

Introduction
Currently, an increasing number of patients with myeloma are
referred for high-dose therapy (HDT), usually 1 or 2 courses of
HDT with an autologous peripheral blood stem cell (PBSC)
support.1 A careful prognostic evaluation of these patients is
warranted to identify patients who maximally benefit from HDT as
well as those who fail current HDT and are candidates for more
innovative treatments. Up to now the best single and routinely
performed prognostic factor remains serum ␤2 microglobulin
(␤2m). Its powerful prognostic significance has been recognized in
many studies over the last 15 years2-6 and confirmed in patients
receiving HDT.7-11 Recently, abnormalities of chromosome 13
(monosomy and/or deletions, ⌬13) have been associated with a
short response duration and survival.12-15 In 2 studies, ⌬13 had been
detected using conventional cytogenetics (CC), which is hampered
by the low mitotic activity of myelomatous plasma cells.12,13 Strong
evidence now indicates that CC fails to recognize about one half of
⌬13 as compared to fluorescence in situ hybridization (FISH), with
incidences of 15% to 20% and almost 40%, respectively.12-17
Recently, 2 studies have used FISH to detect ⌬13 and have
demonstrated its adverse prognostic value, but these patients had
received conventional chemotherapy.14,15 This study was designed
to reevaluate the prognostic value of ⌬13 detected by the FISH
technique and to attempt to define a new prognostic staging for
patients referred for HDT.
Patients, materials, and methods
Patients
The study included 110 patients with multiple myeloma diagnosed in the
centers of Lille (85 patients) and Nantes (25 patients) between May 1990
and September 1998. All patients received an intensive treatment according
to IFM protocols (mainly IFM 94 and 95), following 4 to 5 cycles of VAD
chemotherapy (vincristine, Adriamycin, dexamethasone). The median time
from diagnosis to HDT was 5.2 months. Total body irradiation was used in
the conditioning regimen in 57 patients. The types of stem cells were bone
marrow in 26 patients, PBSCs in 74 patients (2 patients with CD34
selection), PBSCs and bone marrow in 5 patients, and no stem cell support
in 5 patients (high-dose melphalan 140 mg/m2 alone). All patients were
evaluable for response. The patient and treatment characteristics are given
in Table 1.

From the Service d’Hématologie and Laboratoire de Génétique Médicale, Lille,
France; Service d’Hématologie and Laboratoire d’Hématologie, Nantes,
France; Laboratoire d’Hématologie, Angers, France; and Equipe
Biostatistiques/Biomathématiques, Université Paris VII, Paris, France.
Submitted December 14, 1999; accepted October 27, 2000.
Supported in part by the Foundation contre la Leucémie, the Comité Départemental
de Loire-Atlantique de la Ligue contre le Cancer, and the CHRU Lille.
Reprints: Thierry Facon, Service des Maladies du Sang, Hôpital Claude
Huriez, 59037 Lille Cédex, France; e-mail: t-facon@chru-lille.fr.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology

1566 BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6

BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
Table 1. Patient and treatment characteristics (n ⴝ 110,
except where indicated)
Parameter at diagnosis No. of patients
Age ⱖ 60 y 33
Stage III 64
PCL 6 5
B substage (n ⫽ 104) 11
IgA isotype (n ⫽ 108) 23
␭ light chain (n ⫽ 108) 36
␤2m ⱖ 2.5 mg/L 79
CRP ⱖ 6 mg/L (n ⫽ 102) 43
Bone marrow plasmacytosis ⬎ 25% (n ⫽ 108) 74
Albumin ⱕ 35 g/L (n ⫽ 108) 30
Type of HDT
HDM 140 alone 5 5
1 autotransplant 62
Tandem autotransplants 20
1 HDT at diagnosis ⫹ 1 autotransplant at first relapse 21
Other type* 2 2
PCL indicates primary plasma cell leukemia; HDT, high-dose therapy; HDM 140,
high-dose melphalan 140 mg/m2.
*One allogeneic bone marrow transplant and one autotransplant followed by an
allogeneic transplant.
Cytogenetic studies
Conventional cytogenetics and FISH were performed as previously de-
scribed.16,18,19 For FISH, we used a D13S319-specific probe, purchased
from Vysis (Voisin-le-Bretonneux, France) according to the manufacturer’s
instructions. This probe maps to the 13q14 region, in the common deleted
region observed in B-cell chronic lymphocytic leukemia (B-CLL). For 44
patients, the analysis was performed on highly purified plasma cells from
fresh bone marrow collected at diagnosis. For 66 patients, the analysis was
performed on frozen cytospin slides of bone marrow collected at diagnosis.
All samples were collected before treatment and 100 cells were analyzed in
both techniques.
For the 44 patients with purified plasma cells from fresh bone marrow,
the cells were fixed, after thawing, in methanol/acetic acid (3:1, v/v) for 30
minutes and air dried. After hybridization with the D13S319 probe and
wash, cells were counterstained with DAPI in antifade, and examined using
an epifluorescence microscope (Axioplan 2, Zeiss, Iena, Germany) equipped
with a charged couple device camera and appropriate filters. According to
previous studies, ⌬13 was defined by the presence of only one fluorescent
signal in at least 6% of plasma cells.16 However, in this series, the lowest
percentage of plasma cells exhibiting only one signal was 72%.
For the 66 patients with unselected cells, but with frozen cytospin
slides, we used the D13S319-specific probe in a FISH method correspond-
ing to interphase FISH coupled to immunologic revelation of the plasma
cells. Briefly, after fixation in methanol/acetic acid (v/v), slides were
incubated with fluorescein isothiocyanate (FITC)–labeled polyclonal antihu-
man light chains (final dilution, 1:100), as previously described.19 After-
ward, the probe was layered on the slides (overnight hybridization). After
washes, slides were counterstained with DAPI and were examined under a
fluorescence microscope (DMRXA Leica, Wetzlar, Germany) using spe-
cific filters. Bone marrow samples from 8 healthy donors (bone marrow
donors) were used as controls and were studied in the same conditions as
those applied for the multiple myeloma patients (with the exception of
immunostaining). The number of cells demonstrating one spot never
exceeded 8% and, using mean plus 2 SDs, monosomy was ascertained
when over 15% cells exhibited one red signal. However, in this study, all
patients had at least 30% plasma cells with one red signal.
Statistical analysis
The following initial parameters were examined for their prognostic value
on survival and progression: age, sex, stage (Durie and Salmon), substage,
bone marrow plasmacytosis, M-component isotype, type of light chain,
creatinine, ␤2m, lactate dehydrogenase (LDH), C-reactive protein (CRP),
DELETION OF 13q14 IN MYELOMA 1567
hemoglobin, leukocytes, platelets, calcium, albumin serum level, CC, and
⌬13 FISH analysis. The distributions of each variable were compared
% between patients with ⌬13 and those without through chi-square method or
30
Wilcoxon test.20 Curves for progression-free survival (PFS), proportion of
58
patients still in remission, survival after progression, and overall survival
(OS) were calculated from diagnosis as time to event (death without
11
progression or progression, progression, death after progression, death,
21
respectively) using the Kaplan-Meier method21 and compared using the
33
log-rank test.22 The estimate of the relative risk (RR) of event and its 95%
71
confidence interval (95% CI) were estimated through the proportional
42
hazard model.23 In these prognostic analyses, continuous variables were
69
categorized in the following way. Each variable was first divided into 3
28
categories at approximately the 33rd and 67th percentiles. If the relative
event rates24 (observed number of events divided by the expected number of
events in a category, assuming no variation of event rate across categories)
56
and the median times to event in 2 adjacent categories were not substan-
18
tially different, these categories were grouped together.25 If no clear patterns
19
were observed, the median and usual limits (as 2.5, 3, 4, and 6 mg/L for
␤2m) were taken as cut points. As a consequence, 2 to 3 categories were
used for each continuous variable. Then, the ability of each parameter to
increase predictive ability of the ␤2m in overall survival was tested through
backward stepwise proportional hazard model23 including as prognostic
factors ␤2m, the other tested parameter, and their interaction. Quality of the
subsequent statistically significant models were classified according to the
decrease in ⫺2 log (likelihood) when compared to the model with no
covariates, a decrease which is approximately distributed as a chi-square in
which degrees of freedom correspond to the number of factors present in the
model,26 the greater corresponding to the best fit for a fixed number of
degrees of freedom. The same procedure was used to improve the
prediction of the model including ␤2m and ⌬13. No further improvements
were looked for according to the number of deaths in our sample. The best
prognostic models derived with 2 or 3 parameters were simplified by using
the same procedure as the one described for univariate analysis leading to
final models with 3 or 4 categories, only according to the number of
unfavorable factors. The comparison of these final models with previously
published models was performed by using Kaplan-Meier estimates of
survival curves and quantified through the decrease in ⫺2 log (likelihood),
calculated on the same sample as explained above. All analyses were
performed with SPSS software.27
Results
After a median follow-up time of 48 ⫾ 3 months among surviving
patients, 79 progressions and 53 deaths, all after progression except
one, were observed. The median PFS time, time to progression,
survival after progression, and OS time for the whole series were
25 ⫾ 2, 25 ⫾ 2, 12 ⫾ 3, and 51 ⫾ 7 months, respectively. Re-
sponse to treatment was no response (⬍ 20% decrease of the
M-component) in 22 patients (20%), minor response (20%-49%
decrease of the M-component) in 10 patients (9%), partial response
(50%-90% decrease of the M-component) in 22 patients (20%),
very good partial response (⬎ 90% decrease of the M-component)
in 33 patients (30%), and complete response (absence of the
paraprotein on immunofixation and ⱕ 5% plasma cells in the bone
marrow) in 23 patients (21%).
⌬13 was found in 42 patients (38%). In addition to the
chromosome 13 FISH analysis, 68 patients had bone marrow
collected for CC. These patients consisted of the majority of
patients from Lille (no CC was performed in Nantes). This
cytogenetic analysis was unsuccessful in 8 patients (12%) and 60
patients had assessable metaphases. Conventional karyotype was
abnormal in 29 patients and 16 of these 29 patients had ⌬13 (results
of CC and FISH analysis were concordant for these patients). In
these latter patients, additional abnormalities besides those of
chromosome 13 were investigated. The number of chromosomes
1568 FACON et al BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6

Table 2. Prognostic factors at diagnosis, univariate analysis

Progression-free Survival after
Overall survival† survival progression
N* RR† P‡ RR P N§ RR P

⌬13 Abn/N 110 3.3 ⬍ .0001 2.2 .0005 79 2.4 .0018

␤2m (mg/L) ⱖ 2.5/⬍ 2.5 110 4.6 .0001 2.3 .0017 79 2.6 .0176
ⱖ 3.0/⬍ 3.0 110 2.2 .0100 1.8 .0164 79 1.4 .2255
Conv karyotype Abn/N 60 3.4 .0005 2.4 .0048 46 2.0 .0530
LDH (UI/L) ⬎ 230/ⱕ 230 98 2.6 .0013 1.5 .0941 77 2.1 .0120
Stage PCL/I-II 110 5.7 .0015 3.5 .0069 79 4.5 .0084
III/I-II 1.8 1.8 1.1
Type of light chain ␭/K 108 2.2 .0048 1.1 .5978 77 2.2 .0052
Age (y) ⱖ 60/⬍ 60 110 2.1 .0126 1.4 .2023 79 1.8 .0439
Hemoglobin (g/dL) ⬍ 10/ⱖ 10 110 2.0 .0131 1.4 .1452 79 1.6 .1252
CRP (mg/L) ⬎ 2.5/ⱕ 2.5 102 2.1 .0187 1.4 .1508 73 1.9 .0513
ⱖ 6.0/⬍ 6.0 102 1.9 .0323 1.2 .4230 73 2.0 .0190
Isotype IgA yes/no 108 1.9 .0381 1.6 .0731 77 1.9 .0384
IgG no/yes 108 1.6 .0753 1.8 .0088 77 1.3 .4174
Creatinine (mg/L) ⬎ 10/ⱕ 10 110 1.7 .0597 1.3 .2646 79 1.7 .0489
Plasmacytosis (%) ⬎ 25/ⱕ 25 108 1.5 .1542 1.7 .0327 77 1.2 .5119
⬎ 33/ⱕ 33 108 1.4 .2674 1.6 .0520 77 1.2 .5633
Albumin (g/L) ⱕ 35/⬎ 35 108 1.5 .1725 1.1 .7723 77 1.6 .1073

* Number of patients involved in overall survival and progression-free survival analyses.

†Relative risk of event.
‡Significance level.
§Number of patients involved in survival after progression analysis.

was less than 46 in 7 patients, equal to 46 in 4 patients, and more
than 46 in 5 patients. Structural abnormalities of chromosome 14
were frequent: add (14)(q32) to 6 patients and t(11;14)(q13;q32) to
3 patients. Deletion 6q was observed in 2 patients and deletion 16q
in 2 patients (1 patient had nullosomy 16 and the other had der16
t(1;16)(p22;q11)).
A significant positive association was found between ⌬13 and
female sex; ⌬13 was observed in 59% of female patients versus
28% of male patients (P ⫽ .008). It was also the case for ⌬13 and
light-chain myeloma; ⌬13 was present in 64% of patients with
light-chain myeloma versus 34% of patients with another isotype
(P ⫽ .030). Furthermore, ⌬13 myelomas tended to have a higher
stage (⌬13 ⫽ 4 of 6 patients with primary plasma cell leukemia,
44% of stage III patients, and 25% of stage I/II patients; P ⫽ .054).
When comparing mean levels (mean ⫾ SE) of continuous items in
patients with ⌬13 versus in patients without ⌬13, patients with ⌬13
had a higher LDH serum level (302 ⫾ 160 versus 255 ⫾ 295 UI/L;
P ⫽ .037), a lower hemoglobin level (10.1 ⫾ 2.0 versus 11.3 ⫾ 1.6
g/dL; P ⫽ .002), older age (56 ⫾ 6 versus 52 ⫾ 8 years; P ⫽ .030),
and tended to have a higher ␤2m level (5.8 ⫾ 5.1 versus 4.0 ⫾ 2.6
mg/L; P ⫽ .092). Nevertheless, some patients had ⌬13 despite low
␤2m; 9 (21% of patients with ⌬13) and 15 patients (36%) had ⌬13
and a serum ␤2m level below 2.5 and 3.0 mg/L, respectively.
In the univariate analysis, the parameters significantly affecting
PFS, survival after progression, and OS are presented in Table 2.
⌬13 was the most powerful adverse prognostic factor for all times
to event. Median survival times for patients with and without ⌬13
were 27 ⫾ 4 and 65 ⫾ 10 months, respectively (P ⬍ .0001).
Median PFS times for patients with and without ⌬13 were 17 ⫾ 2
and 33 ⫾ 4 months, respectively (P ⫽ .0005) (Figures 1A and 2A).
The response to the initial cytoreductive therapy (VAD) was not
related to OS and PFS (P ⫽ .15 and P ⫽ .30, respectively) nor the
type of bone marrow (fresh versus frozen) for FISH ⌬13 analysis
(P ⫽ .204 and P ⫽ .935, respectively). In our sample, which
included 14 patients with stage I disease who received HDT
according to IFM group criteria, no differences in OS and PFS
could be seen between stage I and stage II patients. Concerning the
23 patients who reached a complete remission, their median
complete remission duration was 28.1 ⫾ 2.8 months (13 patients
with progression). No factors reached statistical significance for
complete remission duration although ⌬13 (P ⫽ .094, RR ⫽ 2.5)
and ␤2m 2.5 mg/L or greater (P ⫽ .094, RR ⫽ 3.4) had a borderline
significance.
In the multivariate analysis the comparison of OS prognostic
models including ␤2m 2.5 mg/L or greater, another factor, and their
interaction is presented in Table 3. In this approach, the decrease in
⫺2 ⫻logarithm (likelihood) makes the comparison possible be-
tween various prognostic models through a unique criterion
distributed as a chi-square. The comparison favored the combina-
tion of ␤2m and ⌬13 (Figure 1B) preceding the combination of ␤2m
and IgA isotype, when associated to ␤2m (Figure 1C). All other
combinations were clearly inferior, including the ␤2m/CRP combi-
nation. Different cut-off values for ␤2m were tested for the
␤2m/⌬13 model (3, 3.5, 4, and 6 mg/L) and the 2.5 mg/L level was
found to be the best cut-off value (data not shown). Patients with no
unfavorable factor (low-risk group), ␤2m less than 2.5 mg/L,
absence of ⌬13 represented 20% of the population (22 of 110) and
had a median survival time more than 111 months (2 deaths among
22 patients). Patients with one unfavorable factor (intermediate-
risk group), 50% of the population (55 of 110), had a median
survival time of 47.3 ⫾ 4.6 months (29 deaths among 55 patients;
RR of death 9.8; 95% CI, 2.3-42.0). These patients consisted of 46
patients with ␤2m 2.5 mg/L or greater and lacking ⌬13 and 9
patients with ␤2m less than 2.5 mg/L and ⌬13. At the time of this
report and taking into account the low number of patients with ␤2m
less than 2.5 mg/L and ⌬13, the OS curves of these 46 and 9
patients seem comparable and similar to that of the entire group (55
patients) (data not shown). Patients with ⌬13 and ␤2m 2.5 mg/L or
greater (high-risk group) represented 30% of the whole population
and had a median survival time of 25.3 ⫾ 3.2 months (22 deaths
among 33 patients; RR of death 27.9; 95% CI, 6.3-124; P ⬍ .0001).
These latter patients had a median PFS time of 15.2 ⫾ 2.0 months.
The results of the ␤2m/⌬13 model regarding OS and PFS are shown
in Figures 1B and 2B. Among the subgroup of 68 patients who had
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
Figure 1. Overall survival according to the number
of unfavorable factors. (A) Chromosome 13 deletions
(⌬13). (B) ⌬13, ␤2m ⱖ 2.5 mg/L. (C) ␤2m ⱖ 2.5 mg/L,
isotype IgA associated to ␤2m ⱖ 2.5 mg/L. (D) ⌬13,
␤2m ⱖ 2.5 mg/L, isotype IgA associated to ␤2m ⱖ 2.5
mg/L. O/N indicates number of deaths per number of
patients. P ⬍ .0001 in all panels.
bone marrow collected for CC, we compared the use of FISH and
CC. Eight patients were unclassified using CC, whereas they were
evaluable using FISH; 7 patients belonged to the intermediate-risk
group (6 progressions, 3 deaths); and 1 patient belonged to the
high-risk group (rapid progression and death). In addition, 7
patients with an apparent normal karyotype using CC had ⌬13
using FISH. Three patients (3 progressions, 2 deaths) would have
been classified in the intermediate-risk group using CC, whereas
they belonged to the high-risk group using FISH. The 4 other
Figure 2. Progression-free survival according to the number of unfavorable
factors. (A) Chromosome 13 deletions (⌬13). (B) ⌬13, ␤2m ⱖ 2.5 mg/L. O/N
indicates number of progressions and deaths without progression, per number of
patients. P ⫽ .0005 in panel A and P ⬍ .0001 in panel B.
DELETION OF 13q14 IN MYELOMA 1569
patients (3 progressions, 2 deaths) switched from the low-risk to
the intermediate-risk group using FISH.
Although inferior to the ␤2m/⌬13 results, the results of the
␤2m/IgA isotype model were interesting and are shown in Figure
1C regarding OS. Furthermore, the following step of the multivari-
ate analysis demonstrated that the OS prediction of the ␤2m/⌬13
model was strengthened by the addition of the IgA isotype to the
model, considering the 3 following parameters: ␤2m 2.5 mg/L or
greater, ⌬13, IgA isotype associated to ␤2m 2.5 mg/L or greater
(Figure 1D). Patients without any adverse factor had a median
survival time more than 111 months (2 deaths among 22 patients).
Patients with 1 or 2 factors had median survival times of 52.3 ⫾ 7.2
and 31.5 ⫾ 4.3 months, respectively (RR of death 8.9; 95% CI,
2.1-38.7 and 23.5; 95% CI, 5.2-106, respectively). This stratifica-
tion seemed particularly useful to isolate a small subgroup of 8
patients with ⌬13, ␤2m 2.5 mg/L or greater and IgA isotype who
had a very poor prognosis; these patients had a median PFS time of
7.8 ⫾ 0.6 months (RR of death without progression or progression
16.1; 95% CI, 6.0-43.3) and a median OS time of 11.7 ⫾ 2.3
months (8 of 8 deaths; RR of death 156; 95% CI, 30-841) (Figure
1D). We finally compared our 2 models, ⌬13/␤2m and ␤2m/⌬13/
IgA, with a previously published prognostic model28 and a
proposed adaptation of this model. We considered 2 ␤2m/CRP
models: ␤2m 2.5 mg/L/CRP or greater 6.0 mg/L or greater and ␤2m
6.0 mg/L/CRP or greater 6.0 mg/L or greater. These comparisons
were done on a group of 102 patients (the whole sample except
nonexcreting multiple myeloma and patients with CRP missing),
and models including ⌬13 appeared clearly superior. The decrease
in ⫺2 log (likelihood) was 52.695 and 35.614 for the ⌬13/␤2m/IgA
and the ⌬13/␤2m models, respectively, but only 21.476 for ␤2m 2.5
mg/L/CRP, 6.0 mg/L or greater, and 14.391 for ␤2m 6.0 mg/L /CRP,
6.0 mg/L or greater.
Discussion
This study emphasizes the pejorative role of FISH ⌬13 on overall
survival, especially in combination with a high serum ␤2m level.
1570 FACON et al
Table 3. Comparison of prognostic models including ␤2m > 2.5 mg/L and another factor*
Factor in addition to
␤2m ⱖ 2.5 mg/L (n ⫽ 110) (n ⫽ 108)
None 18.465 18.269
Chr 13 deletion 35.643 35.620
Age ⱖ 60 y 22.842 22.782
Creatinine ⬎ 12 mg/L 22.076 21.818
Isotype IgA 32.148
␭ light chain 24.503
CRP ⱖ 6 mg/L
LDH ⬎ 230 UI/L
Sample Any patient Except non-excreting
MM
*Only relevant combinations are presented. The higher the decrease in ⫺2 ⫻ logarithm (likelihood), which follows a chi-square distribution with 2 degrees of freedom
(except in line “non” with one degree of freedom), the more powerful is the corresponding model.
The studied sample was composed of 44 patients diagnosed
between January and September 1998, representing the patients of
both centers subsequently treated by HDT, patients for whom FISH
⌬13 analysis was performed on fresh bone marrow. The other 66
patients were a subsample of the patients diagnosed between May
1990 and December 1997 and then treated by HDT in the Lille
center (20-30 patients per year), for whom frozen cytospin slides
performed at diagnosis were still available in 1998 when the
laboratory in Nantes started routine FISH ⌬13 analysis. Therefore,
the selection bias was very unlikely. Indeed, no differences could
be demonstrated between those 2 groups of patients in overall
survival (P ⫽ .204) or PFS (P ⫽ .935). No differences could be
demonstrated between the survival of the 2 groups among patients
with a good, intermediate, or poor prognosis (defined by zero, one,
or 2 adverse prognostic factors, ␤2m and ⌬13). Similarly, differ-
ences between the 2 subsamples were limited to the date of
diagnosis, age (mean 3 years higher in patients diagnosed in 1998),
bone marrow plasmacytosis, and albumin level (mean 12% and 3
g/L higher in the same group).
Chromosome 13 abnormalities were analyzed using a commer-
cial probe mapping at 13q14, containing the D13S319 locus. This
locus appears to be included in the most frequently deleted region
as defined in the recent study of Shaughnessy and coworkers.29
Nevertheless, there is now evidence that marginal differences may
occur with the use of different probes. In future studies it would
probably be of interest to use more than one probe and carefully
study, in a large group of patients, the clinical outcome with respect
to different sites of deletion.
Recently Barlogie and colleagues have recognized ⌬13 as a
highly unfavorable prognostic factor for event-free survival, OS,
and complete response duration, but the study was plagued by the
use of CC and the incidence of ⌬13 was only 16%.13 FISH
techniques are far more efficient than CC to identify numerical
chromosomal abnormalities in myeloma30-32 and indeed we ob-
served a 38% incidence of ⌬13. This incidence was comparable to
that found in the recent FISH study reported by Perez-Simon and
colleagues involving 63 patients who received a standard treatment
(33%) and allows a more precise evaluation of ⌬13 prognostic
value.17 In our study the superiority of FISH over CC was
established in the subgroup of 68 patients who had both analyses.
In our hands, the presence of ⌬13 was critically important for all
times to event, especially PFS and OS; patients lacking ⌬13 had a
median survival of approximately 65 months compared to 27
months for patients with ⌬13 (P ⬍ .0001). Nevertheless, patients
with ⌬13 tend to achieve complete remission more frequently,
possibly reflecting more pronounced proliferative features, higher
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
Decrease in ⫺2 ⫻ logarithm (likelihood)
(n ⫽ 102) (n ⫽ 100) (n ⫽ 90)
15.384 15.202 10.950
33.767 34.768 27.736
18.300 18.169 13.303
18.953 18.707 14.301
30.260 24.214
20.232 15.646
21.477 21.262 17.734
17.313
Except CRP missing Except non-excreting MM Except non-excreting MM,
and CRP missing CRP and LDH missing
sensitivity to HDT, but rapid relapse. To a certain extent, the role of
prognostic factors appeared to vary with the time to event
considered. Whereas ⌬13 and ␤2m were related to all times to event
studied, the type of light chain, age, CRP, and creatinine were
mainly related to survival and plasmacytosis to progression.
Similarly, the IgA isotype was more related to survival and IgG to
progression (Table 2). Such results could explain some variations
observed in prognostic factors from one study to another, due to
particularities of the corresponding sample. Only a suggestive
correlation was found between ⌬13 and ␤2m serum level, which
was so far the best reported prognostic factor in myeloma.2-6 Nine
of 31 patients (29%) with ␤2m serum level below 2.5 mg/L had
⌬13. These patients had a survival time clearly inferior to that of
good-risk patients with low ␤2m and no ⌬13. They would have
been wrongly classified as good-risk patients in the absence of ⌬13
FISH analysis, illustrating further the strong contribution of this
latter analysis to the prognostic classification.
In this study the multivariate analysis for OS prognosis was
performed with the use at the first step of ␤2m (Table 3) and not
⌬13, which was the most significant factor in the univariate
analysis. We did that to determine the contribution on survival of
factors in addition to the ␤2m level, so far the best prognostic factor.
Nevertheless, when using classical stepwise approach, ⌬13 was
introduced at the first step and ␤2m at the second step, leading to the
same bivariate model as the one presented in Table 3. In any case
the IgA isotype was selected at the third step. Overall, whatever the
method used, the 3 significant factors in the multivariate analysis
were ⌬13, ␤2m, and IgA isotype. We agree that in the latter model
the highly pejorative group consisted of a small number of patients
(8 patients) and this result would have to be confirmed in another
sample. Nevertheless, this model with 4 prognostic categories
defined by ␤2m, ⌬13, and IgA isotype was highly significant when
compared to the model with 3 prognostic categories defined by ⌬13
and ␤2m level, and the survival time of this subgroup was actually
very short with a median OS time of approximately 12 months.
Concerning the main end-point, OS, besides the effect of
␤2m alone, several interesting interrelationships have been
published, which permitted the stratification into clinically
useful categories. In particular, the stratifications according to
␤2m and serum albumin,33 ␤2m and CRP,28 and ␤2m and plasma
cell labeling index34 have been found pertinent. In this study,
the ⌬13/␤2m model was far superior to ␤2m/CRP models. The
addition of serum albumin, at the cut-off value of 35 g/L, did
not improve the model containing ␤2m (the cut-off value of 30
g/L could not be used, because only 6 patients had a lower
serum level). The plasma cell labeling index was not available
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6 DELETION OF 13q14 IN MYELOMA 1571

in the vast majority of our patients and therefore this model
could not be considered. In contrast, a good stratification was
achieved using ␤2m and IgA. The adverse prognostic value of
the IgA isotype in the HDT setting has already been pointed
out.9 The ␤2m/IgA model was less powerful than the ⌬13/␤2m
model (Table 3). Good-risk patients had a slightly inferior
survival and the high-risk group consisted of only 14 patients
(13%), whereas there were 33 (30%) in the ⌬13/␤2m model
(Figure 1B,C). Nevertheless, the ␤2m/IgA model appeared as a
valid alternative to the ⌬13/␤2m model for investigators lacking
cytogenetics.
We conclude that ⌬13, adequately detected by FISH analysis, is
a very strong factor related to poor PFS and OS, especially when
associated with ␤2m 2.5mg/L or greater. Although we have no real
explanations concerning the mechanism by which ⌬13 results in
such an adverse prognosis, we strongly recommend routine FISH
⌬13 assessment for patients considered for HDT. More needs to be
done to improve HDT, especially for patients with ⌬13 and high
␤2m who unequivocally fail current HDT, including tandem
autotransplants. For these patients, our group has decided to
evaluate a new tandem transplant program, using melphalan 200
mg/m2 with the first HDT cycle and, depending on age and
availability of an HLA-identical sibling, melphalan 220 mg/m2 and
a monoclonal anti-interleukin 6 antibody, with a second autotrans-
plant or a nonmyeloablative allogeneic stem cell transplantation
(for patients ⬍ 60 years old with an HLA-identical sibling).
Thalidomide, which has recently demonstrated a strong antitumor
activity in refractory myeloma,35 also deserves a complete and
urgent evaluation.
Acknowledgments
We would like to thank Colette Geneix, Jacqueline Regnault, and
Myriam Cambié for their excellent technical assistance.

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