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.     

PCR analysis of immunoglobulin heavy chain and TCR gene rearrangements in diagnosis of lymphoproliferative disorders on formalin-fixed, paraffin-embedded tissues

To confirm a diagnosis of malignant lymphomas it is imperative to distinguish between reactive and neoplastic proliferation. The PCR (polymerase chain reaction) is a method that can be used for detection of clonal rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes. This study summarizes the outcomes of PCR analysis of IgH and TCR gene rearrangements in 91 bioptic cases of lymphoproliferative disorders. In the class of B lymphomas we detected clonal IgH rearrangement in nearly 83% of cases and in class of T lymphomas in 81% of cases. We can affirm that PCR analysis of B and T cell clonality on DNA extracted from the whole section of formalin-fixed, paraffin-embedded tissue is very suitable for routinely elaborate this. Its influence on the diagnostics of morphological unclear cases in particular, is crucial and is useful in establishing a diagnosis of lymphoid neoplasias in specimens in which histological and immunophenotypic studies are inconclusive.     

Molecular and clinical follow-up after stem cell transplantation for multiple myeloma

 Molecular follow-up has been carried out using immunoglobulin heavy-chain (IgH) gene fingerprinting, a polymerase chain reaction (PCR)-based technique with a sensitivity of 0.1–0.01% (10^–3–10^–4), in 22 patients affected by multiple myeloma and submitted to stem cell transplantation (SCT). Twelve patients were submitted to either single or double autologous unselected peripheral blood progenitor cell transplantation, eight patients were submitted to autologous CD34+ immunoselected transplantation and two patients were submitted to allogeneic bone marrow (one patient) or peripheral blood CD34+ stem cell (one patient) transplantation. At diagnosis, all patients showed clonal CDIII rearrangement. The molecular analysis performed on leukapheresis products and CD34+ purified fractions proved to be contaminated by myeloma cells. During follow-up after autografting, all but one patient retained clonal rearrangement despite clinical complete remission (CR) in ten of them. These ten patients either relapsed (Rel) or showed progressive disease (PD) after transplantation; four of them died. Only one patient did not retain clonal rearrangement after autologous transplantation; she is currently alive in CR after a follow-up of 100 months. One patient submitted to allogeneic transplantation is currently alive with no evidence of the disease, but still retains clonal rearrangement after a follow-up of 47 months. Another patient died 4 months after transplantation after succumbing to fatal pneumonia showing myeloma progression. Received: 28 February 2000 / Accepted: 1 August 2000     

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。     

Immunoglobulin and T cell receptor beta chain gene rearrangement analysis of ocular adnexal lymphoid neoplasms: clinical and biologic implications

We investigated the organization of the immunoglobulin heavy chain (IgH) and the T cell receptor beta chain (T beta) gene loci in 20 ocular adnexal and four extraocular lymphoid neoplasms obtained from 18 patients presenting with an ocular adnexal lymphoid neoplasm. Fifteen ocular adnexal and four extraocular lymphoid neoplasms occurring in 13 patients were classified by morphological examination and immunophenotypic analysis as monoclonal B cell lymphomas. Each one of these 19 lymphoid neoplasms exhibited clonal IgH gene rearrangements upon hybridization of EcoRI- or HindIII-digested DNA to a heavy-chain joining region (JH)-specific DNA probe. The bilateral ocular adnexal monoclonal B cell neoplasms occurring simultaneously in two individuals exhibited identical clonal IgH gene rearrangements, which indicated their derivation from an identical B cell clone. The ocular adnexal and the extraocular monoclonal B cell neoplasms occurring in two of three patients also exhibited identical clonal IgH gene rearrangements, which suggested that they too were derived from an identical B cell clone. Five ocular adnexal lymphoid neoplasms were classified by morphological examination and immunophenotypic analysis as benign, polyclonal pseudolymphomas. Three of these five ocular adnexal lymphoid neoplasms exhibited clonal IgH gene rearrangements, which suggested the presence of monoclonal B cell populations that escaped detection by morphological and immunophenotypic examination. None of the 24 pathological samples exhibited clonal T beta gene rearrangements upon hybridization of EcoRI- or BamHI-digested DNA to a T beta gene DNA probe. The results of these studies demonstrate the value of Southern blot hybridization analysis for clonal IgH and T beta gene rearrangements in the diagnosis, classification, and investigation of extranodal lymphoid neoplasms originating and/or presenting in the ocular adnexa.     

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.     

Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B-cell lymphoma and leukaemia: a novel strategy and its implications

In the search of B-cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi-nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B-cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (P cns L) ( n =10) or secondary (S cns L) ( n =7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B-cell population was confirmed in 3/10 of suspected P cns L, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B-cell population in the clinical context of an autoimmune disorder. Evidence of clonal B-cell population was found in 4/7 of suspected S cns L. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B-cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.     

The use of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for quantitative evaluation of multiple myeloma

BACKGROUND AND OBJECTIVES Fluorescent molecular beacons have been employed as hybridization probes in real time quantitative PCR to quantify residual disease in multiple myeloma (MM). DESIGN AND METHODS: After clinical diagnosis of MM, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. A molecular beacon probe for the beta-globin gene was used as a reference control to calculate relative amounts of the clonal B-cell population. RESULTS: Optimization of probe design resulted in the use of a competitive sequence at the IgH area target between the loop and part of the stem of the molecular beacon. Cycling conditions and fluorescence temperature acquisition were optimized for a Light Cycler. To validate this method for the follow-up of treated MM patients, we investigated accuracy, as well as interassay and intrassay reproducibility. CONCLUSIONS: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM.     

Chronic myeloid leukaemia lymphoid blast crisis. Relevance of molecular analysis at the bcr and immunoglobulin heavy chain gene level in monitoring response to therapy and residual disease

Southern blot analyses were performed in sequential DNA samples from 4 patients with Ph' + chronic myeloid leukaemia (CML) who underwent lymphoid or mixed blast crisis (BC). Genomic rearrangements at the breakpoint cluster region (bcr) and immunoglobulin heavy chain (IgH) gene level provided, in these cases, a sensitive and specific evaluation of response to therapy both in terms of blasts and Ph' + cell suppression. Recurrent BC was molecularly characterized in the 4 patients, showing each time identical individual specific DNA rearrangement patterns. Residual blasts were detected in 2 cases during intervening chronic phases by IgH rearrangements. Such findings highlight the specificity of these molecular markers, clearly indicating the failure of ablative therapy in eradicating the neoplastic clone. Finally, molecular and phenotypic identity in individual recurrent BC also suggested, in our cases, a lack of clonal evolution during disease progression.     

Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas

Follicular lymphomas comprise almost two thirds of the US adult non- Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR- amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.     

Molecular immunoglobulin/T-cell receptor clonality diagnosis by gene scan in lymphoproliferative disorders

There remains significant controversy over the techniques used for clonal diagnosis of lymphoproliferative disorders because of questions regarding the sensitivity, specificity and throughput. This has stimulated us to explore the use of gene scan to determine clonality of Immunoglobulin (Ig)/T-cell receptor (TCR) (gamma gene rearrangement in a variety of morphologically, cytochemically, pathologically and immunophenotypically defined precursor B/T-ALL (12 patients), 5 patients with NHL, 10 patients with CLL and a group of reactive lymphocytosis as a reference group (10 subjects). Polymerase chain reaction (PCR) was done for IgH gene (FR3a, FR2b, LJH and JH primers) and for TCR gamma gene and the malignant clone was identified using gene scan (GS) analysis. In the ALL group, monoclonality was detected using GS and using IgH (FR2b) 75% had a clonal band, 63% with IgH (FR3a) and 88% with a combination of FR3a/FR2b. The results of TCR gamma monoclonality were 50% using primer mix I, 25% using primer mix II and 75% using a combination. In the CLL group, clonal IgH gene rearrangement was detected by FR2b in 80% of cases, while by FR3a clonal rearrangement was detected in 60%, the combination of FR2b and FR3a increased the detection rate to 90%. In B-cell NHL, the FR2b was clonally rearranged in 50% while FR3 was positive in 25%. In reactive lymphocytosis, all cases revealed polyclonal rearrangement with TCR gamma primers. The sensitivity and positive predictive value of GS was 100% and the specificity and negative predictive value was 86.6%. In conclusion, gene scanning provides a sensitive and specific method for detection of the malignant clone in PCR product in patients with lymphoproliferative disorders.     

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.     

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.     

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).     

脾边缘带淋巴瘤的临床及肿瘤细胞特征的研究

目的 提高对脾边缘带淋巴瘤（SMZL）的认识和诊疗水平,方法 报告1例典型SMZL,外周血,骨髓及脾脏标本分别采用光镜,相差显微镜,扫描电镜,免疫组化染色,流式细胞术,G显带核型分析及PCR技术研究其肿瘤细胞的生物学特征。结果 本例患者肿瘤细胞为B淋巴细胞,不伴有绒毛,表达CD20,HLA-DR、CD45RA和bcl-2,无异常核型,肿瘤细胞主要浸润脾脏白髓致边缘带明显扩大,脾门淋巴结受累,骨髓和外周血与脾脏有相同的单克隆IgH重排基因,治疗7个月后转为多克隆重排,结论 脾大,外周血或骨髓淋巴细胞比例增高而无淋巴结肿大和白细胞增高患者应疑及SMZL,单克隆IgH基因重排有助于SMZL的诊断,对可疑病例应尽早切脾以明确诊断及防止恶性转化。     

Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma

In an attempt to define the clinical utility of immunoglobulin heavy chain (IgH) gene rearrangement identification for tumour cell detection in multiple myeloma, we investigated 36 consecutive newly diagnosed patients intended for high-dose chemotherapy in a study protocol. After identification of the IgH rearrangement, an allele specific oligonucleotide (ASO) was constructed and used in a semiquantative PCR for minimal residual disease (MRD) evaluation. The myeloma-specific IgH gene rearrangement could be identified and an ASO primer constructed in 24 (67%) of the patients. All of these patients underwent transplantation; 22 were autologous, of whom three had PCR-negative stem cell harvests, and two were allogeneic. 10 patients achieved a clinical complete response (CR) and five were PCR negative in sequential bone marrow analyses. In patients not achieving CR, PCR negativity was occasionally found, but in general the PCR results reflected the clinical status of the patients. No consistent relationship between the bone marrow MRD status and the clinical course was found, and early relapses occurred also in PCR-negative patients.     

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.     

Wotherspoon criteria combined with B cell clonality analysis by advanced polymerase chain reaction technology discriminates covert gastric marginal zone lymphoma from chronic gastritis

BACKGROUND AND AIMS: Gastric mucosa associated lymphoid tissue lymphoma is a well defined B cell lymphoma yet often impossible to distinguish from severe chronic gastritis on morphological grounds alone. Therefore, it was suggested to use the clonality of the immunoglobulin (Ig) heavy chain (H) genes, as detected by polymerase chain reaction (PCR), as a decisive criterion. However, there is controversy as to whether B cell clonality also exists in chronic gastritis, hence rendering this approach futile at present. METHODS: An expert panel re-examined the histology and immunohistochemistry of a total of 97 cases of gastric biopsies, including clearcut marginal zone lymphoma, chronic gastritis, and ambiguous cases, applying the Wotherspoon criteria on the basis of haematoxylin-eosin and CD20 immunostainings. In addition, a new and advanced PCR system for detection of clonal IgH gene rearrangements was independently applied in two institutions in each case. RESULTS: The overall IgH clonality assessments of both institutions were in total agreement. Overt lymphoma (Wotherspoon score 5) was clonal in 24/26 cases. Chronic gastritis (Wotherspoon scores 1 and 2) was not clonal in 52/53 cases; the clonal case being Wotherspoon score 2. Of 18 cases with ambiguous histology (Wotherspoon scores 3 and 4) four were clonal. CONCLUSIONS: Using advanced PCR technology, clonal gastritis is extremely rare, if it exists at all. Thus B cell clonality in Wotherspoon 3 and 4 cases is regarded as suitable for definitively diagnosing gastric marginal zone lymphoma.     

Linear reduction of clonal cells in stem cell enriched grafts in transplanted multiple myeloma

In 30 patients with multiple myeloma who were scheduled for peripheral blood stem-cell transplantation, a quantitative analysis of the stem cells following enrichment by anti-CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele-specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem-cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi-nested PCR combined with a real-time quantitative PCR. In order to accomplish a statistically adequate end-point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34-enriched fraction. The CD34 selection procedure resulted in a median 28-fold enrichment of CD34+ haemopoietic precursor cells. The stem-cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).