Community respiratory virus infections in patients with hematologic malignancies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.     

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.     

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.     

Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis

The rearrangement patterns of Ig and T-cell receptor (TcR) genes were studied by Southern blot analysis in 30 precursor B-cell acute lymphoblastic leukemias (B-ALLs) and 10 T-ALLs at diagnosis and subsequent relapse. Eight precursor B-ALLs appeared to contain biclonal/oligoclonal Ig heavy-chain (IgH) gene rearrangements at diagnosis. Differences in rearrangement patterns between diagnosis and relapse were found in 67% (20 cases) of precursor B-ALLs (including all eight biclonal/oligoclonal cases) and 50% (five cases) of T-ALLs. In precursor B-ALLs, especially changes in IgH and/or TcR-delta gene rearrangements were found (17 cases), but also changes in TcR-beta, TcR- gamma, Ig kappa, and/or Ig lambda genes (11 cases) occurred. The changes in T-ALLs concerned the TcR-beta, TcR-gamma, TcR-delta, and/or IgH genes. Two precursor B-ALLs showed completely different Ig and TcR gene rearrangement patterns at relapse, suggesting the absence of a clonal relation between the leukemic cells at diagnosis and relapse and the development of a secondary leukemia. The clonal evolution in the other 23 ALL patients was based on continuing rearrangement processes and selection of subclones. The development of changes in Ig and TcR gene rearrangement patterns was related to remission duration, suggesting an increasing chance of continuing rearrangement processes with time. These immunogenotypic changes at relapse occurred in a hierarchical order, with changes in IgH and TcR-delta genes occurring after only 6 months of remission duration, whereas changes in other Ig and TcR genes were generally detectable after 1 to 2 years of remission duration. The heterogeneity reported here in Ig and/or TcR gene rearrangement patterns at diagnosis and relapse might hamper polymerase chain reaction (PCR)-mediated detection of minimal residual disease (MRD) using junctional regions of rearranged Ig or TcR genes as PCR targets. However, our data also indicate that in 75% to 90% of ALLs, at least one major rearranged IgH, TcR-gamma, or TcR-delta band (allele) remained stable at relapse. We conclude that two or more junctional regions of different genes (IgH, TcR-gamma, and/or TcR-delta) should be monitored during follow-up of ALL patients for MRD detection by use of PCR techniques. Especially in biclonal/oligoclonal precursor B-ALL cases, the monitoring should not be restricted to rearranged IgH genes, but TcR-gamma and/or TcR-delta genes should be monitored as well, because of the extensive changes in IgH gene rearrangement patterns in this ALL subgroup.     

High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing

The distinction between benign polyclonal and malignant monoclonal lymphoid disorders by morphology or immunophenotyping is frequently difficult. Therefore, the demonstration of clonal B-cell or T-cell populations by detecting identically rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is often used to solve this diagnostic problem. Whereas the detection of rearranged Ig genes is well established, TCR gamma (gamma) and beta (beta) gene rearrangements often escape detection with the currently available polymerase chain reaction (PCR) assays. To establish a sensitive, specific, and rapid method for the detection of rearranged TCR-beta genes, we developed a new PCR approach with family-specific Jbeta primers and analyzed the resulting PCR products by high-resolution GeneScan technique. The superior efficiency of this new method was demonstrated by investigating 132 DNA samples extracted from lymph node and skin biopsy specimens (mostly formalin fixed) and blood samples of 62 patients who had a variety of T-cell lymphomas and leukemias. In all but 1 of the tumor samples (98.4%) a clonal amplificate was detectable after TCR-beta PCR and the same clonal T-cell population was also found in 15 of 18 (83%) of the regional lymph nodes and in 7 of 11 (64%) of the peripheral blood samples. Direct comparison of these results with those obtained currently by the most widely applied TCR-gamma PCR revealed an approximate 20% lower detection rate in the same set of samples than with the TCR-beta PCR method. These results indicate that the new TCR-beta PCR is most suitable for a rapid and reliable detection of clonal T-cell populations. (Blood. 2000;96:640-646)     

Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are assumed to be unique 'fingerprint-like' sequences for each acute lymphoblastic leukaemia (ALL). Various clonal Ig/TCR gene rearrangements can be identified at diagnosis in virtually all childhood ALL patients, representing molecular targets for detection of minimal residual disease (MRD) during follow-up analysis. The usage of at least two MRD-PCR targets per patient generally ensures high sensitivity (相似文献   

Rapid detection of clonality in patients with acute lymphoblastic leukemia

BACKGROUND AND OBJECTIVES: Polymerase chain reaction (PCR) detection of clonal T-cell receptor (TCR) gamma and delta gene rearrangements is widely used in clonality assessment of lymphoid leukemias and lymphomas and for detection of minimal residual disease of acute lymphoblastic leukemia (ALL). Standard analyses for clonality assessment include Southern blotting or PCR-based detection of clonal TCR gene rearrangements. The latter consist of heteroduplex PCR analysis by separation of PCR products on non-denaturing polyacrylamide gel (PAGE). We describe a rapid and sensitive method to identify specific clonal rearrangements in PCR fragments obtained by amplification of TCRgamma and TCRdelta genes. DESIGN AND METHODS: We applied a semi-automated electrophoretic technique (PhastSystem , Amersham Pharmacia Biotech) and compared it with standard homo-heteroduplex analysis in 21 cases of childhood acute lymphoblastic leukemia (ALL). RESULTS: The results obtained for each sample analyzed by standard homo-heteroduplex detection were completely reproduced by the PhastSystem approach. INTERPRETATION AND CONCLUSIONS: We conclude that heteroduplex analysis of TCR gene rearrangements using the semi-automated PhastSystem is a simple, rapid, cheap and highly reproducible method which can be used as an alternative to traditional analysis for detection of clonality.     

Clonal rearrangements in childhood and adult precursor B acute lymphoblastic leukemia: a comparative polymerase chain reaction study using multiple sets of primers.

Ig heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements were investigated by polymerase chain reaction (PCR) amplification of diagnostic tumour samples from 91 patients (57 children and 34 adults, with cut-off at age 16) with precursor B acute lymphoblastic leukemia (ALL). Using primers directed to the framework regions (FR) 1, 2 and 3 of the IgH gene, clonal IgH rearrangements were observed in 82, 58 and 58%, respectively, whereas clonality was presented in 45 and 27% using primers hybridising to the TCR delta and gamma genes. A combination of all five primer sets used resulted in 96% positive cases (children 100%, adults 88%). The frequency of clonal IgH rearrangements correlated to patient age with a significantly lower fraction of positive cases in the adult group. The concomitant usage of more than one V(H) family gene was similar for childhood and adult ALL, and an over-representation of V(H)6 rearrangements was found in childhood ALL. Twenty-five out of 91 cases (27%) displayed an oligoclonal pattern for either IgH or TCR gene rearrangements (children 37%, adults 12%). A comparative analysis of samples from different compartments was performed in 23 patients, and differences between two or three compartments were observed in seven cases. Unexpectedly large, clonally appearing PCR products of 540-715 bp were found in three leukemias and sequence analysis verified their clonal nature. In summary, using multiple sets of primers clonal rearrangements of IgH and TCR genes can be detected in a very high frequency, including previously neglected large size PCR products. A common heterogeneity was demonstrated in different compartments reflecting ongoing clonal evolution, which can make detection of minimal residual disease (MRD) in ALL troublesome. Therefore, we suggest that a minimum of three targets should be used to minimise false-negative results.     

Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B-ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecular studies in 96 childhood precursor-B-ALL at diagnosis and at relapse (n = 91) or at presumably secondary acute myeloid leukemia (n = 5). Clonal Ig and TCR targets for MRD detection were identified in 94 patients, with 71% of these targets being preserved at relapse. The best stability was found for IGK-Kde rearrangements (90%), followed by TCRG (75%), IGH (64%), and incomplete TCRD rearrangements (63%). Combined Southern blot and PCR data for IGH, IGK-Kde, and TCRD genes showed significant differences in stability at relapse between monoclonal and oligoclonal rearrangements: 89% versus 40%, respectively. In 38% of patients all MRD-PCR targets were preserved at relapse, and in 40% most of the targets (> or = 50%) were preserved. In 22% of patients most targets (10 cases) or all targets (10 cases) were lost at relapse. The latter 10 cases included 4 patients with secondary acute myeloid leukemia with germline Ig/TCR genes. In 5 other patients additional analyses proved the clonal relationship between both disease stages. Finally, in 1 patient all Ig/TCR gene rearrangements were completely different between diagnosis and relapse, which is suggestive of secondary ALL. Based on the presented data, we propose stepwise strategies for selection of stable PCR targets for MRD monitoring, which should enable successful detection of relapse in most (95%) of childhood precursor-B-ALL.     

Follow-up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome

Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:10(4) to 1:10(5) cells, depending on the patient's rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long-term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.     

B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL

Minimal residual disease (MRD) detection using quantification of clone-specific Ig or TCR rearrangements before and after transplantation in children with high-risk ALL is an important predictor of outcome. The method and guidelines for its interpretation are very precise to avoid both false-negative and -positive results. In a group of 21 patients following transplantation, we observed detectable MRD positivities in Ig/TCR-based real-time quantitative PCR (RQ-PCR) leading to no further progression of the disease (11 of 100 (11%) total samples). We hypothesized that these positivities were mostly the result of nonspecific amplification despite the application of strict internationally agreed-upon measures. We applied two non-self-specific Ig heavy chain assays and received a similar number of positivities (20 and 15%). Nonspecific products amplified in these RQ-PCR systems differed from specific products in length and sequence. Statistical analysis proved that there was an excellent correlation of this phenomenon with B-cell regeneration in BM as measured by flow cytometry and Ig light chain-kappa excision circle quantification. We conclude that although Ig/TCR quantification is a reliable method for post transplant MRD detection, isolated positivities in Ig-based RQ-PCR systems at the time of intense B-cell regeneration must be viewed with caution to avoid the wrong indication of treatment.     

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.     

Results of minimal residual disease (MRD) evaluation and MRD-based treatment stratification in childhood ALL

The study of minimal residual disease (MRD) as a 'surrogate' marker of molecular response to treatment has drawn great interest because of the potential of tailoring treatment and the possibility of gaining insight into the nature of a cure. Polymerase chain reaction-based (PCR-based) detection of MRD by immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements can be applied in more than 90-95% of cases of childhood acute lymphoblastic leukaemia (ALL). Accordingly, several retrospective studies of MRD in childhood ALL have used one of the different PCR approaches for the detection of antigen-receptor gene rearrangements. The promising results on the predictivity of MRD evaluation at the end of induction treatment has raised the need of a new definition of remission. Until now, most PCR-based MRD studies have used semiquantitative methods for the detection of Ig and TCR gene rearrangements. The introduction of real-time quantitative PCR (RQ-PCR) has resulted in the improvement of sensitivity and specificity and has given better quality control of the MRD data. There is an urgent need to incorporate MRD data in clinical studies, properly designed to address treatment questions. In this context several ongoing co-operative study groups have adopted an MRD-based risk group classification to explore whether a better tailored treatment would result in further improvement in cure rates for children with ALL.     

Spontaneous monoclonal immunoglobulin-secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M-Ig) production by circulating cells has not been reported. Using a two-colour ELISPOT assay, Ig-secreting cells (Ig-SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M-Ig in serum was greater than that of the other Ig-SCs. MM patients presented an excess of circulating heavy-chain (alpha or gamma) Ig-SCs (0.38% of the PBMC) with kappa or lambda light chains (0.48%) compared with the number of cells secreting the other heavy- (0.02%) and light-chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the mu-heavy-chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M-Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig-SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta-2 microglobulin concentration (r = 0. 69; P < 0.0003). The number of M-Ig-SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.     

Assessment of clonal evolution at Ig/TCR loci in acute lymphoblastic leukaemia by single-strand conformation polymorphism studies and highly resolutive PCR derived methods: implication for a general strategy of minimal residual disease detection

Junctional sequences of immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements are used as patient-specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high-resolution PCR-based analyses (including denaturing gel electrophoresis and single-stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V7-J7 and V6j-J6i rearrangements were analysed in 12 T-ALLs. V_H-J_H, V7-J7, V62-D63 and, in selected cases, DH-JH rearrangements were studied in 14 B-lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T-ALLs and in 5/14 B-lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient-specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.     

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.     

Characterization of clone-specific rearrangement T-cell receptor gamma- chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing

The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T- NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T- cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.     

Transitions in CD45 isoform expression indicate continuous differentiation of a monoclonal CD5+ CD11b+ B lineage in Waldenstrom's macroglobulinemia.

Waldenstrom's macroglobulinemia (WM) has been hypothesized to be a pleomorphic B-cell malignancy with persistent maturation towards plasma cells in all lymphoid tissue. This proposal is based on detection of a heterogeneous density of monoclonal Ig on peripheral blood B-cells in patients with WM. We now present data derived from 2- and 3-color immunofluorescence and flow cytometric analysis that strongly supports this hypothesis. Abnormally high numbers of B lineage cells, defined by expression of CD19, CD20, and CD24, were found among peripheral blood mononuclear cells (PBMC). These B-cells are monoclonal as defined by light chain expression and by the existence of rearranged Ig genes (Southern blot analysis), although they exhibit heterogeneity in the density of surface light chain. Unlike normal PBMC B-cells, the monoclonal B-cells bear CD5 and CD10 (CALLA), express adhesion and adhesion-related molecules (CD11b, CD9), and appear to be actively differentiating during the course of the disease, based on the pattern of CD45 isoform expression. At any given point in time, the population of monoclonal B-cells is heterogeneous in differentiation stage based on transitions in the expression of CD45 isoforms from expression of CD45RA, the high molecular mass isoforms of CD45, to the low molecular mass isoform CD45R0 which appears only on very late stage B-cells and early plasma cells. For one patient, analysis of CD45 isoform expression over 2 years showed that the monoclonal B-cell population as a whole progressed towards terminal differentiation as defined by loss of CD45RA and acquisition of CD45R0. This indicates a continuously differentiating lineage of an unusual B-cell phenotype, and/or malignant transformation of a distinct lineage of B-cells in WM.