Haematologica 2003 - Supplements

strategy for the detection of rearranged immunoglobulin heavychain
genes (IgH). Nested PCR proved to be sensitive and
specific, detecting up to 10 -5 /10 -6 tumor cells in bone marrow
samples (1).
Recently, molecular monitoring of minimal residual disease
(MRD) after allo-HSCT has been proposed as a prognostic
parameter potentially helpful to take clinical decisions regarding
the immune-suppression tapering or the timing of donor
lymphocyte infusions. However, its effective role in the clinical
setting is still a matter of debate. Thus, we have recently taken
advantage of our sensitive qualitative PCR based approach to
address the issue of the prognostic value of MRD monitoring in
myeloma patients receiving myeloablative allo-HSCT. In an
EBMT retrospective study, we have longitudinally analyzed
MRD status in 48 myeloma patients in complete clinical
remission for whom a specific molecular marker was identified.
Sixteen of 48 patients (33%) remained persistently negative
during the follow up (NEG group). Thirteen of 48 (27%) were
persistently positive (POS group), while the remaining 19
subjects had a mixed PCR response (i.e. detection of alternatively
positive or negative PCR results during the follow up, MIX
group). The actuarial risk of relapse at 5 years in the NEG, MIX
and POS groups was 0%, 33% and 100% respectively. Patients
with persistent PCR-negativity or mixed PCR results had a better
relapse free survival than those who had never reached PCR
negativity (p=0.0001 and p=0.002 respectively). Although the
retrospective selection of the patients has limited the possibility
to correlate MRD results with clinical parameters, we could not
find any clinical feature significantly associated with the
molecular outcome.
Taken together, all these data suggest the existence of a graft
versus myeloma effect and outline the potential role of such an
effect in maintaining a long term remission in allografted
patients. Thus, MRD monitoring could be used as an indirect
parameter of malignant clone control by effector cells of the
donor immune system. Furthermore, our results about patients
experiencing mixed PCR results could reflect the ongoing
balance between graft versus tumor and tumor immune escape.
These findings prompted us to set up a more accurate technique
to evaluate molecular disease in order to better understand a
dynamic situation. Although extremely sensitive, qualitative
monitoring of MRD by nested PCR, does not provide information
about the amount of residual tumor load, and does not show the
kinetics of its possible elimination by the immune system.
To address this issue, recently real-time quantitative PCR
methods (TaqMan PCR) have been used for MRD monitoring (2,
3). We are thus evaluating quantitative MRD analysis of tumor
specific IgH rearrangements in multiple myeloma patients by two
different strategies:
the first strategy relies on a panel of family specific consensus
probes annealing to the framework region 2 or the framework
region 3 of the rearranged IgH genes. In this case the specificity
for the patient sequence is obtained using primers derived from
complementarity-determining regions (4).
the second strategy, which is more time consuming and
expensive, relies on the design of patient specific probes; the
major advantages of this approach are the sensitivity and the
independence from the ratio of hypermutation of the IgH
sequence.
TaqMan protocols that we are currently using can detect up to 10
copies of patient specific IgH rearrangements diluted in 200 ng of
polyclonal genomic DNA. However, since it has not been
clarified yet, neither the reproducibility of quantitative data
among different laboratories, nor the sensitivity of the technique,
which can be influenced by the specific primer and probe
combination, in this phase we are performing the experiments in
duplicate with standard nested PCR to check reliability and
sensitivity of the quantitative approach. Our preliminary data
indicate that a TaqMan PCR based approach is feasible for
monitoring patients with multiple myeloma after allogeneic
HSCT. In figure 1, we report the quantitative MRD monitoring
in a representative patient who reached the molecular remission
after myeloablative allo-HSCT. In this case the progressive
decrease of tumor specific DNAs was concomitant to the
occurrence of chronic GVHD. The other two patients we have
investigated by TaqMan PCR have never obtained a molecular
response. Nevertheless in one case, quantitative MRD monitoring
allowed the detection of a progressive increase in the number of
tumor genomes 10 months prior to the clinical relapse.
In conclusion, our results prompt the use of quantitative methods
to assess the kinetics of graft versus myeloma effect and suggest
that these new techniques of molecular monitoring may be used
to evaluate the tumor burden in order to develop an
“individualized” post-transplant immunotherapy, reducing the
GVHD risk and optimizing the chance of response.
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