Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes

Linke, Burkhard; Bolz, I; Fayyazi, Afshin; Hofen, M von; Pott, Ch; Bertram, J; Hiddemann, Wolfgang; Kneba, Michael

doi:10.1038/sj.leu.2400736

Cited by 58 publications

(47 citation statements)

References 5 publications

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“…The size and profile of the PCR products was determined using GeneScan Analysis software v. 2.1 (Applied Biosystems). 16,17 For the second method (ABI 3100), 1 l of a 5ϫ dilution of PCR products was added to 10 l of a MilliQ:rhodamine-labeled internal standard (GeneScan-500 ROX; Applied Biosystems) mixture (40:1). After denaturation at 95°C for 2 minutes and cooling, the samples were sizeseparated and detected.…”

Section: Heteroduplex and Genescan Analysesmentioning

confidence: 99%

BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics

Sandberg

Gastel-Mol

Verhaaf

et al. 2005

The Journal of Molecular Diagnostics

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To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and Tcell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B-and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. In most patients with suspected lymphoproliferative disorders, discrimination between reactive and malignant cell populations can be assessed by histomorphology or cytomorphology supplemented with immunohistochemistry or flow cytometric immunophenotyping. However, in 5 to 10% of patients, diagnosis is more complicated and less straightforward. In such cases, molecular gene rearrangement studies have proved useful as an additional diagnostic tool. Molecular clonality analysis is based on the fact that, in principle, all cells of a malignancy have a common clonal origin and show clonally (identically) rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes. The diagnosis of malignant B-and T-cell proliferations is therefore supported by the finding of Ig/TCR gene clonality, whereas reactive lymphoproliferations show polyclonally rearranged Ig/TCR genes. 1 Gene rearrangement analysis can be performed by Southern blot (SB)-and polymerase chain reaction (PCR)-based techniques. Despite the high reliability of SB analysis, it is increasingly replaced by PCR techniques because of the greater efficiency and sensitivity of PCR. Moreover, PCR is relatively easy, less labor intensive, and requires much less high-molecular-weight DNA. Also, SB analysis cannot be performed on paraffinembedded tissue because the isolated DNA is often degraded. Therefore, there is a strong need to replace SB analysis with reliable PCR techniques. However, PCR studies have often suffered from false-negative results due to improper annealing of primers and/or the presence of somatic hypermutation. 2 Both SB and PCR analyses of the immunoglobulin heavy chain (IGH) locus have been demonstrated to be very useful and reliable techniques in clonality assessment of suspected B-cell malignancies. However, the most useful gene target for identifying T-cell clonality is less well e...

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Section: Heteroduplex and Genescan Analysesmentioning

confidence: 99%

BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics

Sandberg

Gastel-Mol

Verhaaf

et al. 2005

The Journal of Molecular Diagnostics

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“…FAM-labeled PCR products were size separated on a high-resolution polyacrylamide gel and laserinduced fluorescence analyzed using an ABI 310 genetic analyzer (Applied Biosystems (ABI) Darmstadt, Germany) as described previously (genescanning). 24,26 Follow-up samples were defined as monoclonal if peaks with identical lengths compared to pretherapeutic samples could be identified. In samples lacking amplifiable IgH-rearrangements, an albumin PCR was performed to check DNA quality.…”

Section: Consensus Igh-pcr and Genescanningmentioning

confidence: 99%

“…12,16,18,24,25 This technique is reported to detect one monoclonal B-cell in 2 Â 10 1 -1 Â 10 3 benign leukocytes. 12,16,18,[26][27][28] Recently, four-color flow cytometry using the characteristic immunophenotype of CLL cells (MRD flow) has been introduced as a sensitive and quantitative tool for MRD detection in CLL, 29 thus improving known flow cytometric techniques. 18,30,31 Dilution studies predicted sensitivities of MRD flow of one CLL cell in 10 4 -10 5 normal leukocytes.…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation

et al. 2004

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The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r ¼ 0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

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“…10,33 Therefore, only tumors in which a monoclonal B cell population was detected by Southern hybridization were entered in this study. Furthermore, a limited number of PCR cycles (25)(26)(27)(28)(29)(30) was performed to minimize amplification of rearranged IgL genes from nonmalignant B cells.…”

Section: Discussionmentioning

confidence: 99%

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin’s lymphoma

et al. 1998

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Minimal residual disease (MRD) detection in B cell non-Hodgkin's lymphoma (NHL) patients has been shown to be possible using the rearranged heavy (IgH) chain gene as a tumor marker. To explore a second independent tumor marker, we used specific PCR primer sets to identify tumor-specific rearranged Ig light chain (IgL) genes. Rearranged IgL genes were amplified from lymphoma DNA by multiplex PCR using separate primer sets for the Ig and the Ig genes. They were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was isolated from at least two independent PCR products. From 12 out of 13 intermediate-and high-grade malignant NHL, PCR products could be obtained with IgL specific primers. PCR products from five NHL were studied in detail by cloning and sequencing. The rearranged IgL genes showed 85-100% homology with their closest germ line counterparts. Intraclonal IgL sequence heterogeneity was studied in five lymphomas and detected in only one. Minimal disease was studied in three patients by PCR, followed by Southern hybridization of the PCR product with a lymphoma-specific oligonucleotide probe, which allowed for detection of lymphoma DNA following 1000-fold dilution. Blood samples from one patient, who is in long-term clinical remission, were negative for the lymphoma-specific rearranged Ig gene. In the second patient the rearranged Ig gene was detected during the first clinical remission, that was followed by a nodal relapse, but not during the second remission, that has been stable for almost 3 years now. The third patient was negative for the rearranged Ig gene in blood samples up to 102 months after diagnosis. Circulating lymphoma cells were detected in blood and bone marrow samples which were negative by morphological and immunological criteria. Our studies show that the rearranged IgL gene can be used as a second independent tumor marker in intermediate-and high-grade malignant NHL.

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Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes

Cited by 58 publications

References 5 publications

BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics

BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin’s lymphoma

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