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Clinical Chemistry 48: 1344-1351, 2002;
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(Clinical Chemistry. 2002;48:1344-1351.)
© 2002 American Association for Clinical Chemistry, Inc.

Validation of Sixteen Leukemia and Lymphoma Cell Lines as Controls for Molecular Gene Rearrangement Assays

Rong Yao1, Steven A. Rich1 and Erasmus Schneidera1

1 Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201.

aAuthor for correspondence. E-mail schneid{at}wadsworth.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Assays for rearrangement of the immunoglobulin, T-cell receptor, bcr/abl, and bcl-2 genes are valuable tools to aid in the diagnosis of leukemias and lymphomas and are now offered by many pathology laboratories. However, there is a lack of well-characterized and validated calibrators and positive controls for these assays. We therefore evaluated 16 readily available leukemia and lymphoma cell lines for their potential use as controls.

Methods: DNA and RNA were isolated from each cell line and analyzed by Southern blot and PCR or reverse transcription-PCR (RT-PCR). Rearrangements in the IgJH, IgJ{kappa}, TcR-ß or TcR-{gamma}, bcr/abl, and bcl-2 genes were detected by commercially available probes and primers. Cell lineages were confirmed by immunophenotyping.

Results: Immunoglobulin and T-cell receptor gene rearrangements were identified in five B- and three T-cell lines, respectively. Two cell lines tested positive for the bcr/abl gene, and one was positive for the bcl-2 gene rearrangement by Southern blot.

Conclusions: The 16 cell lines studied can be used as positive controls in molecular detection assays for gene rearrangements. The parallel processing of these cell lines with clinical samples can serve to quality control the experimental procedures from the first step of DNA or RNA extraction to the final step of result analysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Advances in molecular biology have created powerful and useful techniques that complement current clinical laboratory and pathology testing within many disciplines, including genetics, oncology, infectious diseases, and blood transfusion (1)(2)(3)(4). During the past decade, assays based on Southern blot and PCR (5)(6)(7) have been applied to oncology, where molecular genetic analysis enables the detection of chromosomal abnormalities to study the correlation of gene rearrangements with standard pathologic evaluation and tumor development (8)(9).

Clinical molecular biology laboratories commonly use molecular analyses to determine clonality of B (10)(11)(12) and T cells (13)(14) in leukemia and malignant lymphoma (5)(15)(16) and to detect the translocations t(14;18) in follicular lymphoma (17)(18)(19) and t(9;22) in chronic myelogenous leukemia (20)(21)(22). In the future, new analytical methods may provide even broader clinical applications of molecular detection in pathologic diagnosis and evaluation (23)(24)(25)(26).

The detection of specific gene rearrangements adds important information to the evaluation and diagnosis of patients and supplements results from cytogenetics and immunophenotyping. However, the detection of clonality and/or gene rearrangements by molecular techniques depends on the visualization of DNA fragments different from those seen in nonmalignant germline DNA and which frequently represent unique molecular events in a small percentage of cells in the total cell population (13)(14)(27). The overall performance and quality of the assay are assessed with control samples that are analyzed in parallel with the patient samples and most commonly consist of previously tested and validated patient specimens. However, the limited amount of such material requires frequent characterization and validation of new positive control samples when the old ones run out. Furthermore, because these samples are generally stored as DNA, they do not allow controlling for the initial steps of DNA or RNA isolation. A readily available and renewable source of cells would greatly simplify some of these issues and promote the universal standardization of these assays.

In this report, we establish the utility of leukemia and lymphoma cell lines as positive controls in molecular gene rearrangement assays. Their gene rearrangement patterns were defined by Southern blot analysis and PCR or reverse transcription-PCR (RT-PCR) using commercially available probes or primers for the immunoglobulin genes IgJH and IgJ{kappa} for B-cell origin, the T-cell receptor genes TcR-ß and TcR-{gamma} for T-cell origin, and bcl-2 for the t(14;18) and bcr/abl for the t(9;22) translocations, respectively. For comparison, the B- and T-cell origins of the cell lines were verified by immunophenotyping using flow cytometry.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
cells and dna and rna preparations
All cell lines were obtained from the American Tissue Culture Collection and cultured as recommended (Table 1 ). DNA was isolated using the Puregene DNA reagent set (Gentra), and RNA was isolated using the RNeasy reagent set (Qiagen). The DNA and RNA concentrations were determined from absorbance measurements at 260 and 280 nm.


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  		Table 1. Summary of the Southern blot analyses using JH probe IGHJ6, J{kappa} probe IGKJ5, TcR-ß probe TCRBC, bcr/abl TransProbe-1, and bcl-2 probe.

southern blot
DNA (10 µg) was digested with the appropriate restriction enzymes and the recommended buffers (Roche Molecular Biochemicals), and was separated in a 0.7% agarose gel by electrophoresis at 100 V for 3 h in Tris-borate-EDTA buffer. The DNA was transferred to nylon membranes (Roche Molecular Biochemicals) overnight by capillary action. The probes were labeled with [{alpha}-32P]dCTP (NEN Life Science Products), using the prime-it II random primer labeling reagent set (Stratagene), and purified by QIAquick column purification (Qiagen). IGHJ6 and IGKJ5 probes for detection of IgJH and IgJ{kappa} gene rearrangements, respectively, were purchased from Dako. TransProbe-1, which detects the Philadelphia chromosome translocation t(9;22) was obtained from Oncogene Science Diagnostics. All control DNA samples and the bcl-2 probe for detection of the t(14;18) translocation were purchased from Oncogene Research products. Each specific probe was hybridized to the membrane-bound DNA by use of Quick hybridization solution (Stratagene) according to the manufacturer’s recommendations. The membrane was then exposed to a phosphorimager screen for detection of bound radiolabeled probe. The fluorescein-labeled TCRBC probe (Dako) was used to detect the TcR-ß gene rearrangement, using the Dako chemiluminescent system with exposure to x-ray film (Kodak) according to the manufacturer’s instructions.

rt-pcr and pcr
cDNA was synthesized with Omniscript reverse transcriptase using 1 µg of RNA and random hexamer primers (Qiagen). PCR was performed with gene-specific PCR master mixtures containing the appropriate primer pairs as supplied by In Vivo Scribe. The following reagent sets were used: cat. no. 105G-24 for bcl-2 gene rearrangement; cat. no. 102G-24 for IgH and TcR-{gamma} gene rearrangements; cat. no. 103G-24 for bcr/abl t(9:22) translocations; and cat. no. 107G-24 for immunoglobulin {kappa} light chain assays. The primers are an integral part of the master mixture in each reagent set, but their sequences are proprietary to In Vivo Scribe and therefore not given in the instructions that accompany the reagent sets. Either 500 ng of DNA or 5 µL of cDNA from the reverse transcription reaction were used as the template and amplified at 94 °C for 45 s (3 min for the first cycle), 55 °C for 45 s, and 72 °C for 1 min (10 min for the last cycle) for 35 cycles. Nested PCR was performed with 5 µL of a 1:100 (for bcl-2) or 1:10 (for bcr/abl) dilution of the first reaction as template. The PCR products were analyzed on a 2% agarose gel at 100 V for 2 h.

immunophenotyping
The cultured cells were washed twice in Dulbecco’s phosphate-buffered saline, pH 7.1, containing 1 g/L NaN3 and suspended at a density of 5 x 106 cells/mL in this buffer. One hundred-microliter aliquots of the cells were then added to individual 12 x 75 mm Falcon 2052 plastic tubes for incubation with fluorescence-labeled antibodies to detect surface cluster of differentiation (CD) markers. The fluorescence-labeled antibodies used in this study were from Becton Dickinson (CDs 3, 4, 8, 19, and 20), Beckman Coulter (CDs 2, 7, TcR{alpha}ß, TcR{gamma}{delta}, 10, and 11c), and Biosource International (CDs s{kappa}, s{lambda}, IgD, and IgM). They comprised markers specific for T cells (CDs 2, 3, 4, 7, 8, TcR{alpha}ß, and TcR{gamma}{delta}) and B cells (CDs 10, 11c, 19, 20, s{kappa}, s{lambda}, IgD, and IgM). Stained cells were washed once in phosphate-buffered saline, suspended, and fixed in 500 µL of phosphate-buffered saline containing 5 mL/L formaldehyde. Fluorescence was detected on a FACScan flow cytometer (Becton Dickinson), and acquired and analyzed using CELLQuest software (Becton Dickinson) run on a Power Macintosh Model 7600/132 (Apple). Negative controls consisted of fluorescence-labeled nonspecific antibodies of the same isotype as the CD-specific antibodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
detection of b- or t-cell lineage
To identify cell lines that represent the B- or T-cell lineage, DNA samples from 13 known B- and T-cell lines, together with control samples, were digested with the restriction enzymes EcoRI, BamHI/HindIII, and XbaI and examined by Southern blot using labeled IGHJ6, IGKJ5, and TCRBC probes (Table 1Up ). The 1020-bp IGHJ6 probe detects rearrangements in the JH segments of the immunoglobulin heavy-chain gene (Fig. 1A ). Compared with placental DNA, which represents the germline configuration (Fig. 1A , lane 13), EcoRI digestion of DNA extracted from RL, Nam, SB, and Raji cells generated novel bands of distinct sizes (Fig. 1A ), whereas the typical 8-kb germline band disappeared in DNA from Daudi cells (Fig. 1A , lane 5). BamHI/HindIII digestion of DNA from D1, RL, Jcam, Nam, Daudi, SB, J45, Jurkat, and Raji cells and XbaI digestion of RL, Nam, Daudi, and SB cells generated various and distinct novel bands, whereas Raji lost the typical XbaI germline band (Fig. 1A , lane 11). When analyzed by PCR (Table 2 ), RL, SB, and Raji cells showed an ~317-bp product with the FR1 primer pair (Fig. 1B ), and Nam, SB, and Raji cells showed an ~104-bp product with the FR3 primer pair (Fig. 1C ), both of which demonstrated rearrangements in IgJH genes. Although the exact sizes are likely to be somewhat heterogeneous among different samples, discrimination of small differences was beyond the resolving power of the agarose gels used. However, all the products were near the expected sizes, as deduced from the positive control, and small differences did not change the interpretation. Several nonspecific bands were also observed with the FR1 primer pair, but they were larger than the expected product range of ~290–350 bp (Fig. 1B , lanes 1, 3, 7, 8, 9, 10, and 12). In this and the other PCR assays, extra bands were frequently obtained with some of the primer pairs provided with the In Vivo Scribe reagent set. These were generally regarded as nonspecific based on the information provided by the company and the supplied controls. However, we were unable to eliminate these bands, despite trying various reaction conditions. Whether future generations of the primers will alleviate this problem remains to be seen. Thus, it is extremely important to include positive controls that give a PCR product in the expected size range.



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  		Figure 1. Southern blot (A) and PCR (B and C) analysis for immunoglobulin heavy chain rearrangements.

(A), 10 µg of DNA from various cell lines was digested with EcoRI, BamHI/HindIII, or XbaI, separated by agarose gel electrophoresis, and transferred to a nylon membrane. Each membrane was then hybridized with a probe for JH as described in Materials and Methods. Lanes: 1, D1; 2, RL; 3, Jcam; 4, Nam; 5, Daudi; 6, CCRF-SB; 7, J45; 8, Jurkat; 9, CEM; 10, HL60; 11, Raji; 12, Molt3; 13, placental; 14, 20% SU-DHL-4. (B), DNA from the same cell lines as in A was analyzed by PCR with primer pair FR1 as provided in the In Vivo Scribe reagent set. Lanes 1–13, same as in A; lane 14, 1% RL; lane 15, 10% control; lane 16, FR1 control. (C), lanes are the same as in B, except that FR3 primers were used. Lanes 1–13, same as A; lane 14, 20% In Vivo Scribe control; lane 15, 10% Nam; lane 16, FR3 In Vivo Scribe control.


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  		Table 2. Summary of PCR analyses using the IgH primers FR1 and FR3; IgK primers; TcR-{gamma} primers 1 and 2; bcr/abl primers 2b, 2c, 3b, 3d, and 3d; and bcl-2 mbr primers.

The 540-bp IGKJ5 probe was used to detect rearrangements in the J{kappa} region of the immunoglobulin {kappa} light chain gene (Fig. 2A ). Compared with placental DNA (Fig. 2A , lane 13), EcoRI digestion of DNA from RL, Nam, SB, and Raji cells produced distinct novel bands, whereas the typical 9.3-kb EcoRI germline band disappeared in Daudi cells. For the BamHI/HindIII restriction digestion, the IGKJ5 probe revealed novel digestion patterns in RL, Nam, Daudi, SB, and Raji cells. XbaI digestion of DNA from Daudi, SB, and Raji cells generated various novel bands, whereas the typical 9.9-kb germline band was lost in DNA from RL and Nam cells. When analyzed by PCR, Nam and SB cells also showed an ~219-bp product with the J{kappa} primers, which represents rearrangements in the J{kappa} locus (Fig. 2B ).



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  		Figure 2. Southern blot (A) and PCR (B) analysis for immunoglobulin light chain rearrangements.

(A), conditions were the same as described in the legend for Fig. 1Up , except that each membrane was hybridized with a probe for J{kappa} as described in Materials and Methods. Lanes: 1, D1; 2, RL; 3, Jcam; 4, Nam; 5, Daudi; 6, J45; 7, Jurkat; 8, Molt3; 9, CEM; 10, CCRF-SB; 11, Raji; 12, HL60; 13, placental; 14, 20% SU-DHL-4. (B), DNA from the same cell lines as in A was analyzed by PCR with primer pair IgK as provided in the In Vivo Scribe reagent set. Lanes 1–13, same as A; lane 14, 10% In Vivo Scribe control; lane 15, IgK control.

The 378-bp TCRBC probe was used to detect rearrangements in the TcR-ß locus of the T-cell receptor genes (Fig. 3A ). Compared with placental DNA, the DNA from J45, SupT1, Jcam, D1, Molt3, and Jurkat cells showed rearranged patterns after EcoRI digestion. After BamHI/HindIII digestion, the TCRBC probe revealed rearranged patterns in SupT1, Molt3, and CEM cells. Furthermore, XbaI digestion of SupT1, D1, Molt3, and Nam DNA also produced rearranged bands. Finally, when analyzed by PCR with TcR-{gamma} primer pair 1, J45, SupT1, Jcam, D1, CEM, and Jurkat cells showed bands at ~60–80 bp that were somewhat heterogeneous in size among the different cell lines but all within the expected size range indicated by the positive control. Similarly, when analyzed with TcR-{gamma} primer pair 2, these cells gave an ~221-bp product, demonstrating rearrangements in TcR-{gamma} genes (Fig. 3 , B and C).



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  		Figure 3. Southern blot (A) and PCR (B and C) analysis for TCR-ß (A) and TCR-{gamma} gene rearrangements (B and C).

(A), conditions were the same as described in the legend for Fig. 1Up , except that each membrane was hybridized with a probe for TcR-ß as described in Materials and Methods. Lanes: 1, J45; 2, SupT1; 3, Jcam; 4, D1; 5, Molt3; 6, CEM; 7, Jurkat; 8, CCRF-SB; 9, Nam; 10, Raji; 11, Daudi; 12, RL; 13, SupB15; 14, placental; 15, 20% Molt3. (B), DNA from the same cell lines as in A was analyzed by PCR with primer pair TcR-{gamma}1 as provided in the In Vivo Scribe reagent set. Lanes 1–14, same as in A; lane 15, 10% In Vivo Scribe control; lane 16, TcR-{gamma}1 control. (C), DNA from the same cell lines as in A was analyzed by PCR with primer pair TcR-{gamma}2 as provided in the In Vivo Scribe reagent set. Lanes 1–14, same as A; lane 15, 10% IVS control; lane 16, TcR-{gamma}2 control.

The differentiation lineages of the leukocyte-derived cell lines were determined from their surface membrane antigens. Using 18 different monoclonal antibodies in groups of 3 against surface markers, immunophenotyping of 15 of the cell lines showed that CCRF-SB, Raji, Nam, Daudi, SupB15, and RL cells were reactive to several B-cell surface antigen markers, whereas D1, Molt3, Jurkat, CEM, Jcam, J45, and SupT1 cells were reactive to T-cell surface antigen markers (Table 3 ). As expected, no such surface markers were detected on MEG and HL60 cells because of their known non-B- or -T-cell origin.


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  		Table 3. Immunophenotyping of the various cell lines, using 18 monoclonal antibodies (in groups of 3) against surface markers.

detection of the T(14;18) translocation
Twelve cell lines were examined for the t(14;18) chromosome rearrangement, using a probe that detects the bcl-2 major breakpoint region (Fig. 4A ). EcoRI digestion of RL, Raji, and HL60 DNA revealed distinct novel bands compared with placental DNA (Fig. 4A , lane 13). Novel bands were also detected after BamHI/HindIII and XbaI digestion of DNA from RL cells. RL was the only cell line that was positive by PCR with the mbr primer pair (Fig. 4B ).



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  		Figure 4. Southern blot (A) and PCR (B) analysis for bcl-2 gene rearrangement t(14;18).

(A), conditions were the same as those described in the legend for Fig. 1Up , except that each membrane was hybridized with a probe for bcl-2 as described in Materials and Methods. Lanes: 1, D1; 2, RL; 3, Jcam; 4, Nam; 5, Daudi; 6, J45; 7, Jurkat; 8, Molt3; 9, CEM; 10, CCRF-SB; 11, Raji; 12, HL60; 13, placental; 14, 20% SU-DHL-4. (B), DNA from the same cell lines as in A was analyzed by PCR with primers for the bcl-2 major breakpoint region as provided in the In Vivo Scribe reagent set. Lanes 1–13, same as A; lane 14, In Vivo Scribe mbr control.

detection of the T(9;22) translocation
To determine whether any of the cell lines carried the rearranged bcr/abl gene, DNA was digested with the enzyme BglII and probed with TransProbe-1 (Fig. 5A ). This probe spans the 5.8-kb major breakpoint cluster region in the bcr gene, thereby detecting the chromosomal rearrangement that produces the chimeric bcr/abl gene seen in chronic myelogenous leukemia patients. Among 15 cell lines tested, 2 cell lines, K562 and MEG, showed rearranged bands after BglII digestion (Fig. 5A , lanes 10 and 11). K562 cells showed an extra band in addition to the three germline bands (Fig. 5A , lane 10), whereas MEG cells showed one extra band and lost the 4.8-kb germline band (lane 11). When RNA was analyzed by RT-PCR, K562 cells showed 171- and 126-bp bands with primer pair 3b (Fig. 5C , lane 10), 356- and 311-bp bands with primer pair 3c (Fig. 5D , lane 10), and a 242-bp product with primer pair 3d (Fig. 5E , lane 10). MEG cells showed a 300-bp and one smaller band with primer pair 3c (Fig. 5D , lane 11), and SupB15 and MEG cells both showed a 371-bp band with primer pair 2c (Fig. 5B , lanes 9 and 11, the weakest of the three bands for MEG). Primer pairs 3b, 3c, and 3d are specific for rearrangements involving the major breakpoint cluster region that produces the p210 gene product, whereas primer pairs 2b and 2c are specific for the rearrangement that produces the p190 gene product. However, no product at the expected size was obtained with primer pair 2b in any of the cell lines tested (data not shown). Several nonspecific bands were observed, especially with primer pair 3c, but they were smaller than expected for the real product (<300 bp; Fig. 5D , lanes 3, 4, 6, 7, 12, and 13).



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  		Figure 5. Southern blot (A) and RT-PCR (B–E) analysis for the bcr/abl gene.

(A), 10 µg of DNA from various cell lines was digested with BglII, separated by agarose gel electrophoresis, and transferred to a nylon membrane. Each membrane was then hybridized with TransProbe-1 for bcr/abl as described in Materials and Methods. Lanes: 1, D1; 2, RL; 3, Jcam; 4, Nam; 5, Daudi; 6, J45; 7, Jurkat; 8, Molt3; 9, SupB15; 10, K562; 11, MEG; 12, CCRF-SB; 13, Raji; 14, HL60; 15, placental; 16, oncogene control K562; 17, 20% oncogene K562. (B), RNA from the same cells as in A was analyzed by RT-PCR with primers for the bcr/abl gene product p190 as provided in the In Vivo Scribe reagent set. Lanes 1–15, same as A; lane 16, In Vivo Scribe p190 control. (C–E), conditions were the same as for B, except that primers for the bcr/abl gene product p210 were used: (C), primer pair 3b; (D), primer pair 3c; (E), primer pair 3d. Lanes 1–15, same as A; lane 16, In Vivo Scribe p210 control; lane 17, 10-4 p210 controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to evaluate commercially available cell lines for their usefulness as positive controls in assays for B- or T-cell clonality and for t(9;22) and t(14;18) chromosomal translocations. Cell lines were considered positive for a specific rearrangement if different restriction patterns were observed with at least two enzymes when compared with the germline control or if more than one novel band was observed in a single restriction enzyme digestion. By these criteria, SB, Raji, Nam, Daudi, and RL cells were positive for immunoglobulin gene rearrangements with both B-cell-specific JH and J{kappa} probes (Table 1Up ), which are widely used in the detection of a monoclonal population of B lymphocytes associated with B-cell malignant lymphoma (28)(29). Their B-cell origin was further confirmed by immunophenotyping using six combinations of three different monoclonal antibodies, each against either B- or T-cell surface markers (Table 3Up ). These B-cell lines, therefore, qualify as positive controls in assays to detect IgJH and/or IgJ{kappa} locus rearrangements associated with B-cell leukemia and lymphoma. When we used similar criteria, the D1, Molt3, and SupT1 cell lines tested positive with the T-cell-specific probe TcR-ß (14)(30) (Table 1Up ), and their T-cell origins were confirmed by immunophenotyping. These cell lines therefore qualify as positive controls for detection of the rearranged TcR-ß gene.

Because PCR-based assays have become the methods of choice for initial analysis, we also analyzed each cell line by this method. For the B-cell analysis, there was an 80% correlation (four of five cell lines; Table 2Up ) between PCR using the FR1 and/or FR3 primers and Southern blot analysis, and 40% using the IgK primers (two of five cell lines; Table 2Up ). For T-cell analysis, three cell lines were positive by Southern blot for TcR-ß and six cell lines were positive by PCR for TcR-{gamma}. Of those, only two, D1 and SupT1, were positive by both assays. These discrepancies were likely attributable to different regions of the IgH and IgK genes and the distinctive TcR-ß and TcR-{gamma} genes being targeted by the respective primers (31) (In Vivo Scribe) and probes (Dako) (28)(29)(32).

Although a positive control for detecting the bcl-2 gene rearrangement, SU-DHL-4, is commercially available as DNA, we have been unable to obtain the cell line. One of the tested cell lines, RL, was positive with both the bcl-2 probe by Southern blot analysis and the mbr primers by PCR, exhibiting the t(14;18) translocation commonly present in follicular lymphomas (17)(18)(19). Thus, RL appears to be the only cell line readily available as a positive control cell sample for monitoring the whole procedure from DNA preparation to detection.

Both K562 and MEG cells are useful as controls for the Philadelphia chromosome translocation because they tested positive with the bcr/abl-specific TransProbe-1 and showed the presence of the bcr/abl gene product p210 (20)(21) by RT-PCR with primer pairs 3b, 3c, and 3d (all three for K562, and only 3c for MEG). With primer pair 2c, MEG and SupB15 cells were both positive for the bcr/abl gene product p190 and therefore can be used as controls for detection of the p190 gene product.

Over the last several years, novel rearrangements have been reported for the bcl-2 (33) and LAZ3 (BCL6) genes (34) in patients with follicular lymphoma, unusual bcr/abl fusion transcripts (e8/a2 and e13/a2) have been reported in patients with Philadelphia chromosome-positive leukemia (35), and B- or T-cell gene rearrangements have been reported in patients with intraocular lymphoma (36). In addition, the application of molecular detection has led to the identification of the t(14;18) translocation in nonlymphoid tissues (37). Furthermore, new technologies, such as nonradioactive PCR-single strand conformational polymorphism analysis and PCR-temporal temperature gradient gel electrophoresis for the detection of T-cell receptor {gamma}-chain gene rearrangements (38)(39) and real-time quantitative PCR for demonstrating B-cell chronic lymphatic leukemia (40)(41) have been developed.

In conclusion, our study validated 16 cell lines as positive controls for commonly used tests for B- and T-cell clonality and for t(9;22) and t(14;18) translocations (Tables 1Up and 2Up ). The commercial availability of the positive cell lines for each test described in this study provides easily accessible routine controls to monitor and evaluate all steps in the respective assays. They may also be useful when new or updated technologies and/or test procedures are developed. In particular, their use could facilitate interlaboratory standardization and proficiency testing for this increasingly important area of clinical analysis. Finally, the ready availability of these cells can further be exploited to determine the analytical sensitivity of a procedure by mixing a "positive" cell line with a "neutral" cell line in various proportions to create dilution series.


   Acknowledgments
 
We are grateful to the Wadsworth Center’s cell culture and immunology core facilities for their help with culturing and immunotyping the cell lines and to the photography department for their help with the figures.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Narayanan S. Concepts, principles, and applications of selected molecular biology techniques in clinical biochemistry. Adv Clin Chem 1996;32:1-38.[Web of Science][Medline] [Order article via Infotrieve]
  2. Kiechle FL. DNA technology in the clinical laboratory. Arch Pathol Lab Med 1999;123:1151-1153.[Web of Science][Medline] [Order article via Infotrieve]
  3. Aziz KJ. Clinical molecular biology: concepts and applications. Adv Clin Chem 1996;32:39-72.[Web of Science][Medline] [Order article via Infotrieve]
  4. Visscher DW, Sarkar FH, Crissman JD. Molecular genetic analysis in the pathologic evaluation of solid tumors: theory and practice. J Clin Lab Anal 1997;11:10-16.[Web of Science][Medline] [Order article via Infotrieve]
  5. Ladanyi M, Wang S. Detection of rearrangements of the BCL2 major breakpoint region in follicular lymphomas. Correlation of polymerase chain reaction results with Southern blot analysis. Diagn Mol Pathol 1992;1:31-35.[Medline] [Order article via Infotrieve]
  6. Mihalov ML, Huber S, Rachwalski E, Price JS, Hosso JM, Dizikes GJ. Detection of clonal immunoglobulin heavy-chain gene rearrangements in cases of suspected lymphoproliferative disorders: comparison of polymerase chain reaction and Southern blot analysis. South Med J 1996;89:39-45.[Web of Science][Medline] [Order article via Infotrieve]
  7. Sabath DE, Wood BL, Kussick SJ. PCR methods for determining B cell clonality. J Mol Diagn 2000;2:217-218.[Free Full Text]
  8. Lust JA. Molecular genetics and lymphoproliferative disorders. J Clin Lab Anal 1996;10:359-367.[Web of Science][Medline] [Order article via Infotrieve]
  9. Cobaleda C, Perez-Losada J, Sanchez-Garcia I. Chromosomal abnormalities and tumor development: from genes to therapeutic mechanisms. Bioessays 1998;20:922-930.[Web of Science][Medline] [Order article via Infotrieve]
  10. Hieter PA, Korsmeyer SJ, Waldmann TA, Leder P. Human immunoglobulin kappa light-chain genes are deleted or rearranged in {lambda}-producing B cells. Nature 1981;290:368-372.[Medline] [Order article via Infotrieve]
  11. Arnold A, Cossman J, Bakhshi A, Jaffe ES, Waldmann TA, Korsmeyer SJ. Immunoglobulin-gene rearrangements as unique clonal markers in human lymphoid neoplasms. N Engl J Med 1983;309:1593-1599.[Abstract]
  12. Kitchingman GR. Immunoglobulin heavy chain gene VH-D junctional diversity at diagnosis in patients with acute lymphoblastic leukemia. Blood 1993;81:775-782.[Abstract/Free Full Text]
  13. Kitchingman GR, Rovigatti U, Mauer AM, Melvin S, Murphy SB, Stass S. Rearrangement of immunoglobulin heavy chain genes in T cell acute lymphoblastic leukemia. Blood 1985;65:725-729.[Abstract/Free Full Text]
  14. Flug F, Pelicci PG, Bonetti F, Knowles DM, 2nd, Dalla-Favera R. T-Cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms. Proc Natl Acad Sci U S A 1985;82:3460-3464.[Abstract/Free Full Text]
  15. Korsmeyer SJ. Immunoglobulin and T-cell receptor genes reveal the clonality, lineage, and translocations of lymphoid neoplasms. Important Adv Oncol 1987;:3-25.
  16. Neale GA, Kitchingman GR. mRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5' end. Nucleic Acids Res 1991;19:2427-2433.[Abstract/Free Full Text]
  17. Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci U S A 1985;82:7439-7443.[Abstract/Free Full Text]
  18. Cleary ML, Smith SD, Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell 1986;47:19-28.[Web of Science][Medline] [Order article via Infotrieve]
  19. Weiss LM, Warnke RA, Sklar J, Cleary ML. Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas. N Engl J Med 1987;317:1185-1189.[Abstract]
  20. Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, de Klein A, Bartram CR, et al. Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature 1983;306:239-242.[Medline] [Order article via Infotrieve]
  21. Stam K, Heisterkamp N, Grosveld G, de Klein A, Verma RS, Coleman M, et al. Evidence of a new chimeric bcr/c-abl mRNA in patients with chronic myelocytic leukemia and the Philadelphia chromosome. N Engl J Med 1985;313:1429-1433.[Abstract]
  22. Kawasaki ES, Clark SS, Coyne MY, Smith SD, Champlin R, Witte ON, et al. Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci U S A 1988;85:5698-5702.[Abstract/Free Full Text]
  23. Meier VS, Rufle A, Gudat F. Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates. Am J Pathol 2001;159:2031-2043.[Abstract/Free Full Text]
  24. Shiokawa S, Nishimura J, Ohshima K, Uike N, Yamamoto K. Establishment of a novel B cell clonality analysis using single-strand conformation polymorphism of immunoglobulin light chain messenger signals. Am J Pathol 1998;153:1393-1400.[Abstract/Free Full Text]
  25. Beaubier NT, Hart AP, Bartolo C, Willman CL, Viswanatha DS. Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction. Diagn Mol Pathol 2000;9:121-131.[Web of Science][Medline] [Order article via Infotrieve]
  26. Hosler GA, Bash RO, Bai X, Jain V, Scheuermann RH. Development and validation of a quantitative polymerase chain reaction assay to evaluate minimal residual disease for T-cell acute lymphoblastic leukemia and follicular lymphoma. Am J Pathol 1999;154:1023-1035.[Abstract/Free Full Text]
  27. Devlin BH, Ratech H, Kaufman R. Inclusion of a sensitivity control to add a quality-control parameter and improve reproducibility in BCR, immunoglobulin, and T-cell receptor gene-rearrangement studies. Diagn Mol Pathol 1994;3:271-274.[Web of Science][Medline] [Order article via Infotrieve]
  28. Beishuizen A, Verhoeven MA, Mol EJ, Breit TM, Wolvers-Tettero IL, van Dongen JJ. Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results. Leukemia 1993;7:2045-2053.[Web of Science][Medline] [Order article via Infotrieve]
  29. Beishuizen A, Verhoeven MA, Mol EJ, van Dongen JJ. Detection of immunoglobulin kappa light-chain gene rearrangement patterns by Southern blot analysis. Leukemia 1994;8:2228-36(discussion 2237–9).[Web of Science][Medline] [Order article via Infotrieve]
  30. Pelicci PG, Knowles DM, 2nd, Dalla Favera R. Lymphoid tumors displaying rearrangements of both immunoglobulin and T cell receptor genes. J Exp Med 1985;162:1015-1024.[Abstract/Free Full Text]
  31. Miller JE, Wilson SS, Jaye DL, Kronenberg M. An automated semiquantitative B and T cell clonality assay. Mol Diagn 1999;4:101-117.[Web of Science][Medline] [Order article via Infotrieve]
  32. van Dongen JJ, Wolvers-Tettero IL. Analysis of immunoglobulin and T cell receptor genes. Part I. Basic and technical aspects. Clin Chim Acta 1991;198:1-91.[Web of Science][Medline] [Order article via Infotrieve]
  33. Wang YL, Addya K, Edwards RH, Rennert H, Dodson L, Leonard DG, et al. Novel bcl-2 breakpoints in patients with follicular lymphoma. Diagn Mol Pathol 1998;7:85-89.[Web of Science][Medline] [Order article via Infotrieve]
  34. Daudignon A, Bisiau H, Le Baron F, Lai JL, Wetterwald M, Galiegue-Zouitina S, et al. Four cases of follicular lymphoma with t(14;18)(q32;q21) and t(3;4)(q27;p13) with LAZ3 (BCL6) rearrangement. Cancer Genet Cytogenet 1999;111:157-160.[Web of Science][Medline] [Order article via Infotrieve]
  35. How GF, Lim LC, Kulkarni S, Tan LT, Tan P, Cross NC. Two patients with novel BCR/ABL fusion transcripts (e8/a2 and e13/a2) resulting from translocation breakpoints within BCR exons. Br J Haematol 1999;105:434-436.[Web of Science][Medline] [Order article via Infotrieve]
  36. White VA, Gascoyne RD, Paton KE. Use of the polymerase chain reaction to detect B- and T-cell gene rearrangements in vitreous specimens from patients with intraocular lymphoma. Arch Ophthalmol 1999;117:761-765.[Abstract/Free Full Text]
  37. Rauzy O, Galoin S, Chale JJ, Adoue D, Albarede JL, Delsol G, et al. Detection of t(14;18) carrying cells in bone marrow and peripheral blood from patients affected by non-lymphoid diseases. Mol Pathol 1998;51:333-338.[Abstract]
  38. Murphy M, Signoretti S, Kadin ME, Loda M. Detection of TCR-{gamma} gene rearrangements in early mycosis fungoides by non-radioactive PCR-SSCP. J Cutan Pathol 2000;27:228-234.[Web of Science][Medline] [Order article via Infotrieve]
  39. Zhu D, Kadin ME, Samoszuk M. Detection of clonal T-cell receptor-{gamma} gene rearrangement by PCR/temporal temperature gradient gel electrophoresis. Am J Clin Pathol 2001;116:527-534.[Abstract/Free Full Text]
  40. Pfitzner T, Engert A, Wittor H, Schinkothe T, Oberhauser F, Schulz H, et al. A real-time PCR assay for the quantification of residual malignant cells in B cell chronic lymphatic leukemia. Leukemia 2000;14:754-766.[Web of Science][Medline] [Order article via Infotrieve]
  41. Lehmann U, Bock O, Langer F, Kreipe H. Demonstration of light chain restricted clonal B-lymphoid infiltrates in archival bone marrow trephines by quantitative real-time polymerase chain reaction. Am J Pathol 2001;159:2023-2029.[Abstract/Free Full Text]




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