Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay

OBJECTIVE: To develop a real-time PCR method, based on the 5'nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM). MATERIALS AND METHODS: The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells. RESULTS: The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow. CONCLUSION: The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.     

Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin's lymphoma is associated with decreased relapse after autologous bone marrow transplantation

In B-cell non-Hodgkin's lymphoma (NHL), as in other B-cell malignancies, clonal rearrangement of the third complementarity determining region (CDR III) of the immunoglobulin heavy chain gene (IgH) provides a useful marker for the detection of minimal residual disease (MRD) after treatment. To determine the clinical utility of IgH polymerase chain reaction (PCR), we analyzed peripheral blood (PB) and bone marrow (BM) samples from 25 patients with NHL with no PCR detectable chromosomal rearrangement who have undergone autologous bone marrow transplantation (ABMT). Patients with histologic bone marrow infiltration at the time of bone marrow harvest were selected for study since this provided us with diagnostic tissue samples. As an initial strategy DNA was amplified using consensus variable (VH) and joining (JH) region primers. In those cases failing to amplify using consensus region primers, PCR was performed using a panel of VH family-specific framework region 1 (FR1) primers. The clonal products were directly sequenced. From the V-N-D region nucleotide sequences, clone specific probes were constructed and used for subsequent detection of MRD. A clonal PCR product could be PCR amplified and directly sequenced in 18 (72%, 90% confidence intervals 54%-86%) of these 25 patients, 8 with diffuse and 10 with follicular NHL. Eight of these 18 patients have relapsed after ABMT. All had detectable lymphoma cells before relapse and the sequence of the CDR III region at the time of relapse was identical to that obtained at the time of ABMT. All 10 patients who remain in complete remission from 18 to 36 months after ABMT had eradication of PCR detectable lymphoma cells after ABMT, although in three patients PCR detectable MRD was detected early after ABMT. We conclude that sequencing and the use of patient specific IgH CDR III oligonucleotides probes provides a simple and highly reliable method to determine the specificity of the IgH PCR technique. The clinical utility of this technique is demonstrated by the finding that eradication of PCR detectable lymphoma cells in these patients is associated with decreased relapse after ABMT (P = .0002).     

Molecular Monitoring of Minimal Residual Disease in Patients with Multiple Myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.     

Molecular monitoring of minimal residual disease in patients with multiple myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.     

Contaminating tumour cells in autologous PBSC grafts do not influence survival or relapse following transplant for multiple myeloma or B-cell non-Hodgkin's lymphoma

Relapsed disease remains a major obstacle following autologous haematopoietic SCT (HSCT) for non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Studies regarding the importance of detectable tumour cells in PBSC collections have been inconclusive. Patients undergoing autologous HSCT for NHL and MM between 2001 and 2006 were enrolled (n=158). PBSC grafts were assessed for clonal IgH CDR3 gene rearrangements using qualitative semi-nested PCR. In comparison to patients with PCR-positive PBSC grafts, patients negative for detectable disease had no improvement in overall survival (OS) or PFS for MM (P=0.91 and 0.91) or NHL (P=0.82 and 0.85). Further, no significant difference in OS was observed between patients with PCR-positive compared with PCR-negative PBSC grafts with aggressive NHL histology (P=0.74) or indolent disease (P=0.29). Patients with contaminating tumour cells in autologous PBSCs do not have worsened OS or PFS in MM or NHL. Tumour cells detected by sensitive molecular methods in PBSC collections may be distinct from cells contaminating marrow and appear to have limited utility in identifying patients with MM and B-cell NHL who would benefit from purging strategies.     

Molecular analysis of patients with relapsed or refractory intermediate-high grade non-Hodgkin's lymphoma with bone marrow infiltration undergoing peripheral blood progenitor cell transplantation.

BACKGROUND AND OBJECTIVES: IgH gene rearrangement studies with a polymerase chain reaction (PCR) technique can detect the persistence of clonal cells at molecular level during the remission phase. This persistence of clonal cells can be used to establish the relationship between minimal residual disease (MRD) and clinical outcome. We have developed a three-step single strand conformational polymorphism PCR strategy which is able to detect clonal B lymphoid cells at a frequency as low as 1 clonal cell in 10(6) normal cells. DESIGN AND METHODS: Twenty patients with intermediate or high-grade B non-Hodgkin's lymphoma (NHL) were evaluated. Patients were pre-treated with a median of two (range 1-4) conventional chemotherapy lines before high-dose cyclophosphamide (HDCY). All patients had their bone marrow (BM) involved by disease (median 10%; range 5-50%). Nineteen patients were offered high-dose therapy followed by peripheral blood progenitor cells (PBPC) autografting. RESULTS: MRD analysis was performed for each patient at the end of conventional chemotherapy and every three months after high dose therapy. All these patients achieved complete response (CR) after high dose therapy (HDT). Six patients relapsed after a median time of 24.5 months. All the studied apheresis samples were positive at the molecular analysis. All 6 patients still positive at the molecular analysis after PBPC autografting relapsed. The remaining 13 patients who were negative maintained CR. INTERPRETATION AND CONCLUSIONS: Whereas the detection of clonal cells in the apheresis samples did not predict an unfavorable outcome, the disappearance of the clonal rearranged band from the BM sample after HDT proved to be a favorable prognostic factor and was associated with long-lasting disease-free status     

Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation

In chronic lymphocytic leukemia (CLL), clonal rearrangement of the immunoglobulin heavy chain locus (IgH) provides a useful marker for the detection of minimal residual disease (MRD) after treatment. At the time of initial presentation, DNA from patients with CLL was polymerase chain reaction (PCR)-amplified using consensus Variable (VH) and Joining (JH) region primers using complementarity determining region III consensus region primers or a panel of VH family-specific framework region 1 (FR1) primers. The clonal product was directly sequenced and patient-specific probes constructed using N region nucleotide sequences. We amplified and sequenced the CDRIII region and designed patient specific oligonucleotide probes for the detection of MRD in 55 of 66 patients (84%, 90% Confidence Intervals (CI): 74% to 90%) with poor prognosis CLL referred for autologous and allogeneic bone marrow transplantation (BMT). To determine the clinical utility of this technique, PCR amplification was performed on patient samples at the time of and following autologous (21 patients) and allogeneic (10 patients) BMT in whom serial bone marrow samples obtained after BMT were available for analysis. We show that the persistence of MRD after BMT is associated with increased probability of relapse. In all cases that have relapsed to date, the IgH CDRII region was identical at the time of initial presentation and at relapse suggesting that clonal evolution of the IgH locus is unusual in this disease. The finding that a significant number of patients remain disease free and with no evidence of PCR-detectable MRD after BMT suggests that high-dose therapy may contribute to improved outcome in selected patients with CLL.     

High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting

Based on preliminary encouraging results in terms of response rate and survival, high-dose chemoradiotherapy has gained considerable interest in the treatment of patients with multiple myeloma (MM). We have evaluated the presence of residual myeloma cells in 15 of 18 patients enrolled in a high-dose sequential (HDS) chemoradiotherapy program followed by autografting. Our analysis has been performed both on bone marrow (BM) and peripheral blood (PB) cell harvests and after autografting. As it has been recently shown that B cells clonally related to malignant plasma cells are detectable in MM patients, we have developed a polymerase chain reaction (PCR)-based strategy to detect both residual B cells and plasma cells using clone-specific sequences derived from the rearrangement of Ig heavy chain (IgH) genes. The complementarity-determining regions (CDR) of IgH genes have been used to generate tumor-specific primers and probes. The constant (C) region usage defined the differentiation stage of residual myeloma cells. We report that plasma cells were detectable in PB and BM cell harvests and after transplantation in all assessable patients, irrespective of disease status. B cells were detectable in a consistent proportion of BM and PB samples at diagnosis, but only in one case at the time of PB and BM cell harvests. These cells became sometimes detectable after transplantation. Whether residual myeloma cells are clonogenic and contribute to relapse is currently unknown, and further investigations are required.     

Minimal residual disease quantification in childhood acute lymphoblastic leukemia by real-time polymerase chain reaction using the SYBR green dye

OBJECTIVE: Clone specific immunoglobulin (Ig) and T-cell receptor (TCR) gene sequences can be used as molecular targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Real-time quantitative PCR (RQ-PCR) with no need for post-PCR processing is an attractive approach for detection and quantification of specific DNA or RNA sequences. In the present study we evaluated a real-time PCR-based technology for MRD quantification in children with precursor-B ALL. MATERIALS AND METHODS: DNA samples from 36 children with newly diagnosed precursor-B ALL were available for molecular analysis. All patients were uniformly treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocols from 1992. A real-time PCR assay was applied for MRD quantification using LightCycler technology and the SYBR green fluorescent dye for detection of clone-specific Ig and TCR gene rearrangements as target sequences. The specificity of the PCR products was verified by melting curve analysis. RESULTS: Thirty-four of the 36 children with precursor-B ALL (94%) displayed at least one clonal Ig heavy chain (IgH) or TCR gene sequence useful as a molecular target. These clone-specific targets were successfully applied for real-time PCR quantification in all but one patient. Melting curve analysis was important for identifying all specific PCR products. In 32 pediatric precursor-B-ALL patients an MRD level >/=10(-3) at day 29 during induction treatment was significantly correlated with later bone marrow relapse (p = 0.0025). CONCLUSIONS: Real-time PCR using clone-specific primers and the SYBR green dye for detection is a feasible technique for identifying patients at risk for relapse. This approach provides an easily applicable tool for detection of IgH/TCR gene rearrangements in the routine setting. Melting curve analysis allowed clear distinction between specific rearrangements and unspecific background signals.     

Clinical and molecular follow-up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of hematopoietic CD34+ stem cells.

BACKGROUND AND OBJECTIVE: Autologous blood stem cell transplantation (ABSCT) using chemotherapy-induced mobilization of peripheral blood stem cells (PBSC) is being increasingly used in the treatment of multiple myeloma (MM). We report the clinical and molecular follow-up of 10 MM patients who underwent autologous stem cell transplantation with peripheral blood selected CD34+ cells, as support therapy following a myeloablative conditioning regimen. DESIGN AND METHODS: The CDR-III coding region of the IgH gene was studied by a) consensus PCR applied to 8 MM patients, or b) by direct sequencing of PCR product generated by family-specific primers in the remaining two patients (who became immunofixation analysis (IF) negative). In this case, two patient-specific primers were generated, thus obtaining a high PCR assay sensitivity and specificity (ASO PCR). RESULTS: Seven patients are alive: 4 of them have serum M protein assessable by IF, while 1 was not a secretor and 2 converted from serum IF positivity to negativity 6 and 12 months after ABSCT. Three patients died: 1 from disease progression and 2 from infective complications during clinical remission. The molecular analysis during the follow-up showed that the bone marrow samples from the two patients who obtained IF negativity were persistently PCR positive for the presence of rearranged CDR-III region. Moreover, despite the remarkable reduction of myeloma burden, a minimal level of residual myeloma cells was still detectable by molecular analysis. INTERPRETATION AND CONCLUSIONS: These results confirm that although positive selection of CD34+ cells markedly reduces the contamination of myeloma cells from apheresis products by up to 3 log, and provides a cell suspension capable of restoring normal hematopoiesis after ablative conditioning regimen, it does not abrogate myeloma cell contamination in most of the apheresis products.     

Polymerase chain reaction-based detection of minimal residual disease in multiple myeloma patients receiving allogeneic stem cell transplantation

BACKGROUND AND OBJECTIVES: Recent advances in the treatment of multiple myeloma (MM) include use of high-dose chemoradiotherapy followed by allografting. Although allografting with bone marrow (BM) or peripheral blood stem cells (PBSC) seems to improve clinical outcome and lengthen survival, only about 50% of patients reach stringently defined complete remission (CR), and most subsequently relapse. We assessed the clinical relevance of minimal residual disease (MRD) in 14 MM patients in CR after allografting with PBSC (6 patients) or BM (8 patients). DESIGN AND METHODS: Among the 30 out of 72 MM patients in our Institute who achieved CR after allografting, 14 had a molecular marker suitable for allo-specific polymerase chain reaction (PCR) analysis.Stringent molecular monitoring was done using clonal markers based upon rearranged immunoglobulin heavy-chain genes. Molecular remission (MCR) was defined as two consecutive negative PCR results. RESULTS: Seven of 14 (50%) molecularly monitored patients, achieved MCR and did not relapse after a median molecular follow-up of 60 months (range 36-120). Median time to obtain first PCR negativity was 12 (BM group) and 6 months (PBSC group), respectively. Of the seven patients (50%) who never achieved MCR, one relapsed. INTERPRETATION AND CONCLUSIONS: In conclusion, 50% of the MM patients in CR studied by us also achieved stringently-defined MCR. MCR was associated with a very low rate of clinical relapse.     

Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma

BACKGROUND AND OBJECTIVES: Several polymerase chain reaction (PCR)-based techniques for tracking minimal residual disease (MRD) in B-lymphoproliferative disorders have been recently proposed. These procedures show significant variation in sensitivity and specificity. We describe an alternative assay based on fluorescent PCR combined with capillary electrophoresis and GeneScan analysis, to identify the monoclonal immunoglobulin heavy chain (IgH) rearrangement in multiple myeloma (MM) and to provide a semi-quantitative evaluation of MRD by limiting dilutions. DESIGN AND METHODS: Different sets of family specific primers derived from the leader region and from the framework-1 of IgH were used, with a unique reverse fluorescent primer JH. The malignant clone was identified by GeneScan and sequenced. Two tumor primers, mapping in the complementarity determining regions CDRII and CDRIII, were designed for each patient. A comparison between the nested-PCR approach and direct fluorescent PCR was performed for three patients in complete clinical remission after autologous or allogeneic bone marrow transplantation. RESULTS: Thirty-six consecutive patients with MM were screened and monoclonality was identified in about 70% of the cases. Molecular MRD evaluation was performed in 18 patients using tumor primers. This method allowed identification of 1 neoplastic cell among 10(4)-10(6) normal cells. In three cases, negative by nested-PCR and agarose gel electrophoresis, gene scanning showed persistence of the neoplastic clone, despite the negativity of the immunofixation. INTERPRETATION AND CONCLUSIONS: Capillary electrophoresis of fluorescent fragments with gene scanning provides a simple, rapid and reproducible method to detect IgH rearrangement and to evaluate MRD. Furthermore, the sensitivity reached is up to 1 log higher than that of the conventional approach with nested-PCR, even though two steps of specificity are maintained.     

实时定量PCR检测B细胞非霍奇金淋巴瘤患者外周血和骨髓IgH基因重排及临床意义

目的:检测B细胞非霍奇金淋巴瘤(B-NHL)患者外周血和骨髓中IgH基因重排并探讨其在临床诊治中的应用。方法:利用SYBR GreenⅠ荧光染料,采用实时定量PCR方法,以IgH基因为标志,对B-NHL患者治疗后采集的15例外周血及10例骨髓的IgH基因重排进行定量分析。结果:15例外周血及10例骨髓均检测到IgH基因拷贝数,外周血和骨髓差异无统计学意义(P>0.05)。结论:实时定量PCR方法对外周血和骨髓中IgH基因重排定量分析,可以作为B-NHL鉴别诊断和随访微小残留病的辅助手段,并对判断疗效、预测复发有一定的临床意义。外周血和骨髓IgH基因拷贝数差异无统计学意义。     

分子信标探针技术用于PCR检测诺卡氏菌SecA1基因

目的将诺卡氏菌属细菌的一段特异性DNA设计成分子信标探针,用于该细菌的PCR检测。方法将诺卡氏菌属细菌、戈登氏菌属细菌及红球菌属细菌菌株分别接种于脑心浸液琼脂培养基分离培养,观察其生长情况,提取菌株DNA作为扩增模板;设计诺卡氏菌属细菌基于secA1基因的特异性分子信标探针,在实时荧光定量PCR反应译体系中加入分子信标探针,PCR产物进行荧光信号检测。结果诺卡氏菌secA1基因经实时荧光定量PCR扩增后可产生阳性荧光信号,红球菌属细菌及戈登氏菌属细菌的secA1基因、阴性对照实验组及空白对照组经实时荧光定量PCR扩增后不产生荧光信号,为阴性。结论 secA1作为看家基因,是用来进行种水平的鉴定及系统进化研究非常理想的靶分子,而分子信标探针技术可以准确、快速、灵敏的进行诺卡氏菌secA1基因检测。     

Immunoglobulin gene ''fingerprinting'': an approach to analysis of B lymphoid clonality in lymphoproliferative disorders

Rearrangement of the immunoglobulin heavy chain (IgH) gene is widely exploited as a marker of B cell lineage and clonality in the pathology of lymphoproliferative disorders. We have developed a simple, polymerase chain reaction (PCR) based method for detecting IgH gene rearrangement which relies on the observation that by using a panel of PCR amplimers specific for each of the six heavy chain variable region families in conjunction with a common joining region amplimer, clonal rearrangement can be detected in over 90% of cases of B lymphoid malignancy. By using radiolabelled amplimers and exploiting the size heterogeneity resulting from independent IgH rearrangement events, we show that high resolution gel electrophoresis can be used to generate a 'fingerprint' representing the spectrum of B cell clonality in complex populations of B lymphocytes. The method effectively scans the entire IgH gene rearrangement repertoire and is capable of detecting rare clonal or oligoclonal B lymphoid cell populations. In normal bone marrow mononuclear cells, clonal IgH rearrangement could be readily detected at a sensitivity of 10(-3). We illustrate the application of the method in assessing the spectrum of B cell clonality occurring in an autoimmune condition. Hashimoto's thyroiditis, and in a malignant B cell disorder, chronic lymphocytic leukaemia. In addition, we explore the potential application of the technique in tracking minimal residual disease and for monitoring clonal evolution in acute lymphoblastic leukaemia.     

Quantitative Assessment of Minimal Residual Disease in Childhood Lymphoid Malignancies Using an Allele-Specific Oligonucleotide Real-Time Quantitative Polymerase Chain Reaction

We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/10(5) cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lymphoblastic leukemia and 3 of non-Hodgkin's lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.     

Various patterns of IgH deletion identified by FISH using combined IgH and IgH/CCND1 probes in multiple myeloma and chronic lymphocytic leukemia

Introduction: Interphase fluorescence in situ hybridization (FISH) can identify submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. In this study, the characteristics and frequency of the IgH deletion identified by interphase FISH were investigated in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). Methods: The study group included 29 patients with MM and eight patients with CLL. Interphase FISH was performed with the IgH dual color, break‐apart rearrangement probe and the IgH/CCND1 dual color, dual fusion translocation probe. Results: The IgH deletion was found in 14% (4/29) of patients with MM and 13% (1/8) of the patients with CLL. Four patients had deletions of the whole or variable region of IgH on the native chromosome 14, whereas one patient had a deletion of the IgH variable region on a der(11)t(11;14). In two patients, the IgH break‐apart FISH showed both patterns with and without IgH deletions. In cases showing the same pattern by IgH break‐apart FISH, the IgH/CCND1 FISH showed different patterns, and vice versa. Conclusion: A variety of patterns of the IgH deletion were identified by interphase FISH using IgH break‐apart and IgH/CCND1 probes in patients with MM and CLL. The results of this study suggest that the integrated information obtained with IgH break‐apart and IgH/CCND1 FISH was needed to interpret FISH results unambiguously.     

Cellular origin and extent of clonal involvement in multiple myeloma: genetic and phenotypic studies

The cellular origin and extent of clonal involvement in multiple myeloma (MM) are controversial. The third-complementarity-determining region (CDR3) of the immunoglobulin heavy chain gene is the target region of V_H replacements and somatic mutations. We analysed the CDR3 sequences of myeloma cells from eight newly diagnosed and three relapsed patients in order to elucidate the target cell of malignant transformation in MM. We also examined the extent of clonal involvement in MM using a CDR3 clone-specific nucleic acid probe. The peripheral lymphocytes from the five MM patients were separated into fractions such as CD34⁺, CD20⁺CD10⁺, CD20⁺CD21⁺, CD20⁺CD19⁻ and CD2⁺ cells. Amplified CDR3 DNAs from these subpopulations were hybridized with the probe specific to each patient's tumour cells. We found no evidence of ongoing V_H replacements or somatic mutations in CDR3 in MM. However, frequent nucleotide mutations in D and J_H segments were observed. Circulating malignant cells were detected in the CD34⁺ and all of the CD20⁺ subpopulations, but not in the CD2⁺ fraction. MM is a neoplasm originating from a B-lineage cell which has already undergone antigendependent selection. Nevertheless, the tumour cells are composed of heterogeneous subpopulations at various stages of differentiation, similar to normal B-lineage cells. Conversely, T cells were not involved in MM. These results imply that there is an analogous developmental pathway between the normal B-lineage cells and the tumour cells of MM.     

Real-time polymerase chain reaction in multiple myeloma: quantitative analysis of tumor contamination of stem cell harvests

OBJECTIVE: Autologous transplantation of bone marrow (BM) and peripheral blood progenitor cells (PBPC) is commonly used for treatment of multiple myeloma (MM). Although both stem cell sources harbor residual clonal cells, a quantitative evaluation of their level of tumor contamination (LTC) still needs to be performed through highly accurate and reproducible approaches. In this study, we used a validated real-time polymerase chain reaction (PCR) strategy to evaluate LTC of BM and PBPC samples obtained from MM patients. MATERIALS AND METHODS: The patients underwent two different mobilization courses (defined as early or late course) following two cycles of cyclophosphamide 5 g/m(2). LTC was evaluated by measuring the number of clonal immunoglobulin heavy-chain rearrangements followed by normalization of samples using the GAPDH gene. RESULTS: Overall, 26 PBPC and 12 BM samples were analyzed. Main results are as follows. 1) PBPC harvests are less contaminated than BM samples taken immediately after each mobilization course (median difference 2.68 logs; range 1.7 to 4.6) (p < 0.0001). 2) LTC of PBPC harvests has only minimal variation among different leukaphereses performed during the same mobilization course (median difference 0.45 logs; range 0.22 to 1.2). 3) No difference was observed among PBPC and BM samples obtained after the late mobilization course as compared to the early mobilization course (median reduction 0.21 logs; range -0.39 to 1.3) (p = 0.84). 4) In PBPC but not in BM samples, there is a clear overestimation of the percentage of plasma cells when flow cytometric evaluation of CD38(bright) cells is compared to real-time PCR results. This suggests that in PBPC, most CD38(bright) cells do not belong to the neoplastic clone. CONCLUSIONS: Real-time PCR using the IgH rearrangement proved an effective tool for monitoring LTC in stem cell harvests from MM patients. The smaller LTC of PBPC harvests supports the role of PBPC as stem cell rescue for MM patients compared to BM cells.