¹Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Correspondence: M Lioznov, E-mail: lioznov@uke.uni-hamburg.de

We are conducting a program of allo-SCT after nonmyeloablative or dose-reduced preparative regimens to treat multiple myeloma (MM) patients.¹ Recent evidence indicates that minimal residual disease (MRD) in BM is a major prognostic factor after this therapy.² Since MRD might often efficiently be treated by donor lymphocyte infusions,³ highly sensitive and efficient monitoring of MRD is crucial in the post transplant period.⁴

Monitoring of molecular remission is most often performed by qualitative PCR with allele-specific oligonucleotides-PCR(ASO-PCR) based on the design of specific primers detecting the IgH rearrangement for the individual patient.⁴ This is, however, a time-consuming procedure, which may not be successful for each patient. Sarasquete et al.⁵ extensively compared sensitivity, specificity and applicability of ASO-PCR with a novel flow cytometry method (FCM). On the basis of their study, the authors concluded that both techniques provide similar prognostic information. The FCM is well accepted in the diagnosis of MRD in MM.^{6, 7} In a recent review, Bataille et al.⁸ focused on phenotypic markers for malignant and normal plasma cells (PC) and confirmed CD138 as a universal PC marker, whereas overexpression of CD56 along with lack of both CD19 and CD45 is typical only for myelomatous PC.

Here, we performed a comparative analysis of three different techniques in the dynamics after allo-SCT regarding their sensitivity to detect MRD: (i) FCM, (ii) patient-specific RQ-PCR (TaqMan) using ASO primers⁹ and (iii) assessment of lineage-specific PC chimerism by real-time PCR based on bi-allelic sequence nucleotide polymorphism.¹⁰

We investigated 69 BM samples from 13 MM-confirmed patients with advanced-stage disease at different time points after allo-SCT following dose-reduced conditioning based on melphalane and fludarabine. All samples were analyzed by FCM using a panel consisting of CD56/CD19/CD45 following the SSClo/CD38+/CD138+ gating strategy (Figure 1) as described by the above authors.^{5, 6, 7, 8} In parallel, 11 samples were also tested by RQ-PCR with patient-specific ASO primers⁹ and for PC-chimerism analysis¹⁰ as we described earlier.

Figure 1.

Gating strategy for identification of myelomatous PC (mPC) in BM of MM patients. On the basis of 4-color approach, (a) shows fluorescence and light scatter of all (min. 500 000) acquired events. R₁ is used to define the CD38⁺/CD138⁺ PC (b). Doublets of gated PC were excluded in R₂ (c). mPC were defined as CD56⁺⁺/CD19^- /CD45^- cells, which were gated from the combined regions of R₁ and R₂ (d).

Full figure and legend (152K)

FCM and ASO primer PCR results correlated very well (r²=0.94; P<0.0001, Spearman range correlation) with all 69 samples both in positive and negative MRD cases (Table 1). Also, we found significant negative correlations between PC chimerism and both FCM and ASO-PCR results (r²=- 0.84 and –0.81, respectively; P<0.0001). None of the 46 samples, which were positive for MRD using FCM and PCR with patient-specific ASO primers demonstrated a full PC chimerism (>99.9% ), although significant variations were observed (range: 0.00–99.9% ). In contrast, 9 of 23 samples with no detectable MRD (by FCM and ASO-PCR) revealed a full-donor chimerism of >99.9% in the PC department, but 14 of 23 samples had PC chimerism 99.9% (98.8–99.9% ).

Table 1 - Frequencies of phenotypical aberrations in PC as detected by FCM vs the results of PC chimerism and ASO-PCR.

Full table

In summary, the present report shows that the prognostic value of FCM is comparable with that of patient-specific ASO-PCR. There was also a good correlation between FCM and PC-chimerism data in all MRD cases, whereas mixed chimerism was not predictive for MRD. Probably, chimerism kinetics would be much more reliable.

On the basis of these data, we conclude that multiparametric FCM is an effective method for monitoring MRD in MM patients after allo-SCT.