Haematologica 2003 - Supplements

(performance status, age and albumin), related to the tumor load
(β2M and BMPC infiltration) and related to the intrinsic tumor
biology (S-phase PC and LDH level). This analysis allows
distributing patients into different groups probably requiring
different treatment. These results claim for more efforts to clearly
define reproducible systems that actually allow the use of
different therapeutic strategies.
7.4 Minimal residual disease
205
Quantitation of the post-transplantation tumor load in
the bone marrow by PCR with allele-specific
oligonucleotide primers is a prognostic parameter in
multiple myeloma
Marleen H.C. Bakkus1, Yasmina Bouko2, Tina Everaert1,
Eva Lipinski3, Diana Samson4, Jane F Apperley4, Kris
Thielemans5, Benjamin Van Camp1, Axel Benner6,
Hartmut Goldschmidt3, Marion Moos3, Friedrich W.
Cremer3
1Department of Hematology-Immunology, VUB, Brussels, 1090,
Belgium; 2Hematology, ULB Erasme, Brussels, 1070, Belgium;
3Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany.; 4Hematology, Hammersmith Hospital,
London, United Kingdom; 5Department of Physiology, VUB,
Brussels, Belgium; 6Department of Biostatistics, German Cancer
Research Center, Heidelberg, Germany
Background: High dose chemotherapy (HDT) with autologous
hematopoietic stem cell transplantation (PBSCT) significantly
improves survival in multiple myeloma (MM) patients. In a
subgroup of MM patients, early relapses occur after HDT and
autologous transplantation. In order to investigate whether the
amount of residual tumor cells in the bone marrow (BM) after
transplantation can predict the duration of response, a quantitative
PCR assay was used to measure tumor load in BM at 3 to 6
months post-transplant.
Patients and Method: Sixty-six patients with MM treated by HDT
followed by autologous PBSCT, either CD34+-selected or
unselected, were included in this study. The myeloma
immunoglobulin heavy chain gene variable sequence was used as
a tumor specific target, and quantitation of the tumor load was
performed by limiting dilution.
Results: The median level of tumor cells in the BM was 0.04% of
the total mononucleated cell fraction at 3 months post-HDT
(n=59), and 0.021 at 6 months (n=32). Using maximally selected
logrank statistics, a post-HDT BM tumor load cutoff value with
respect to progression-free survival (PFS) was identified
(p=0.001). The threshold for placing patients into a good or bad
prognostic group was found to be 0.015%. Median PFS was 61
vs. 15 months For multivariate analysis, Beta2-microglobulin at
diagnosis, number of high-dose therapy cycles (HDT), best
response after PBSCT (CR versus PR/MR) and grouping by
qPCR result were included in a Cox proportional hazard model as
potentially prognostic parameters. Grouping by PCR result was
found to be an independent and strong predictor of PFS (hazard
ratio, 3.54).
This study demonstrates for the first time a threshold of the post-
HDT tumor load with prognostic significance regarding PFS in
MM. Quantitative molecular assessment discriminates between
low and high risk groups of patients as early as 3 to 6 months
after autologous transplantation, and thus helps to identify those
patients who are in need of further treatment after autologous
transplantation.
206
UNMUTATED INCOMPLETE DJH
REARRANGEMENTES AS PREFERENTIAL TARGET
FOR REAL-TIME PCR QUANTIFICATION IN MULTIPLE
MYELOMA.
González D, González M, Alonso ME, Balanzategui A,
López-Pérez R, Chillón C, García-Sanz R, and San Miguel
JF.
Hematology Department, University Hospital of Salamanca and
Centro de Investigación del Cáncer (CIC), University of
Salamanca, Salamanca, Spain.
The hypervariable regions of immunoglobulin heavy chain (IgH)
rearrangements provide a specific tumor marker in multiple
myeloma (MM). Recently, real-time PCR assays have been
developed in order to quantify the number of tumor cells after
treatment. However, these strategies are hampered by the
presence of somatic hypermutation in VDJH rearrangements
from MM patients, which causes mismatches between primers
and/or probes and the target, leading to a non accurate
quantification of tumor cells. Our group has recently described a
60% incidence of incomplete DJH rearrangements in MM
patients, with no or very low rate of somatic hypermutation. In
this study, we compare the efficiency of a real-time PCR
approach for the analysis of both complete and incomplete IgH
rearrangements in eight MM patients using only three JH
consensus probes. We were able to design an allele specific
oligonucleotide for both the complete and incomplete
rearrangement in all patients. DJH rearrangements fulfilled the
criteria of effectiveness for real-time PCR in all samples (i.e. no
unspecific amplification, detection of less than 10 tumor cells
within 105 polyclonal cells and correlation coefficients of
standard curves higher than 0.98). By contrast, only three out of
eight VDJH rearrangements fulfilled these criteria. Further
analyses showed that the remaining five VDJH rearrangements
carried three or more somatic mutations in the probe and primer
sites, leading to a dramatic decrease in the melting temperature.
These results support the use of incomplete DJH rearrangements,
instead of complete somatically mutated VDJH rearrangements
for investigation of minimal residual disease in multiple
myeloma.
207
Quantification of minimal residual disease by real-time
IgH-PCR using TaqMan chemistry together with
LightCycler technology predicts relapse in multiple
myelom
R. Fenk, G. Kobbe, C. Arnold, U. Steidl, A. Hünerlitürkoglu,
U.P. Rohr, T. Emde, A. Bernhard, R. Haas, R. Kronenwett
Dept. of Hematology, Oncology and Clinical Immunology,
University of Duesseldorf, Germany
Background: In multiple myeloma (MM) PCR with allelespecific
oligonucleotides (ASO) for immunoglobulin H (IgH)
provides a method for detection of minimal residual disease
(MRD). Real-time PCR using LightCycler technology offers the
opportunity to quantify MRD. In general, two hybridization
probes are used for specific detection of the product during realtime
PCR. Since the PCR products in IgH-PCR are too small for
two internal hybridization probes we developed an IgH-PCR
using one patient-specific ASO probe with TaqMan chemistry
adapted for the LightCycler system.
Methods: Patient-specific PCR conditions were established using
ASO primers and an ASO TaqMan probe. Values of absolute
copy numbers derived from an external standard curve with
S179