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Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

2007; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês

10.2353/jmoldx.2007.060149

ISSN

1943-7811

Autores

Jaron VanDijken, Govind V. Kaigala, Jana Lauzon, Alexey Atrazhev, Sophia Adamia, Brian J. Taylor, Tony Reiman, Andrew R. Belch, C. Backhouse, Linda M. Pilarski,

Tópico(s)

Advanced biosensing and bioanalysis techniques

Resumo

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system. Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system. In multiple myeloma (MM), a cancer characterized by extensive, complex chromosomal abnormalities, recurrent translocations involving the immunoglobulin heavy chain gene on chromosome 14 are among the most frequent translocations, being found in about 60% of myeloma patients1Keats JJ Maxwell CA Taylor BJ Hendzel MJ Chesi M Bergsagel PL Larratt LM Mant MJ Belch AR Pilarski LM Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.Blood. 2005; 105: 4060-4069Crossref PubMed Scopus (145) Google Scholar and many myeloma cell lines.2Bergsagel PL Chesi M Nardini E Brents LA Kirby SL Kuehl WM Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma.Proc Natl Acad Sci USA. 1996; 93: 13931-13936Crossref PubMed Scopus (320) Google Scholar The t(4;14)(p16;q32) reciprocal translocation, which results in aberrant regulation of genes on both chromosomes 4 and 14, has been particularly well studied.3Richelda R Ronchetti D Baldini L Cro L Viggiano L Marzella R Rocchi M Otsuki T Lombardi L Maiolo AT A novel chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma involves the fibroblast growth factor receptor gene.Blood. 1997; 90: 4062-4070Crossref PubMed Google Scholar,4Chesi M Nardini E Lim RS Smith KD Kuehl M Bergsagel PL The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts.Blood. 1998; 92: 3025-3034Crossref PubMed Google Scholar As a result of the t(4;14) translocation, the fibroblast growth factor receptor 3 (FGFR3) gene in chromosome 14 is overexpressed, whereas on chromosome 4, theMMSET gene is overexpressed.4Chesi M Nardini E Lim RS Smith KD Kuehl M Bergsagel PL The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts.Blood. 1998; 92: 3025-3034Crossref PubMed Google Scholar Multiple breakpoints on chromosome 4 have been identified within the MMSET gene,3Richelda R Ronchetti D Baldini L Cro L Viggiano L Marzella R Rocchi M Otsuki T Lombardi L Maiolo AT A novel chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma involves the fibroblast growth factor receptor gene.Blood. 1997; 90: 4062-4070Crossref PubMed Google Scholar with REII-BP as a likely target gene.1Keats JJ Maxwell CA Taylor BJ Hendzel MJ Chesi M Bergsagel PL Larratt LM Mant MJ Belch AR Pilarski LM Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.Blood. 2005; 105: 4060-4069Crossref PubMed Scopus (145) Google Scholar In addition, each MM patient is characterized by unique molecular signature, the IgH VDJ gene rearrangement, which provides a unique marker to identify all malignant cells in each patient. Patients having the t(4;14) translocation have reduced survival.1Keats JJ Maxwell CA Taylor BJ Hendzel MJ Chesi M Bergsagel PL Larratt LM Mant MJ Belch AR Pilarski LM Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.Blood. 2005; 105: 4060-4069Crossref PubMed Scopus (145) Google Scholar,5Keats JJ Reiman T Maxwell CA Taylor BJ Larratt LM Mant MJ Belch AR Pilarski LM In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression.Blood. 2003; 101: 1520-1529Crossref PubMed Scopus (335) Google Scholar6Winkler JM Greipp P Fonseca R t(4;14)(p16.3;q32) is strongly associated with a shorter survival in myeloma patients.Br J Haematol. 2003; 120: 170-171Crossref PubMed Scopus (11) Google Scholar7Moreau P Facon T Leleu X Morineau N Huyghe P Harousseau JL Bataille R Avet-Loiseau H Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy.Blood. 2002; 100: 1579-1583Crossref PubMed Scopus (278) Google Scholar t(4,14) myeloma, characterized by drug resistance and rapid relapse,8Jaksic W Trudel S Chang H Trieu Y Qi X Mikhael J Reece D Chen C Stewart AK Clinical outcomes in t(4;14) multiple myeloma: a chemotherapy-sensitive disease characterized by rapid relapse and alkylating agent resistance.J Clin Oncol. 2005; 23: 7069-7073Crossref PubMed Scopus (80) Google Scholar has been shown to predict for poor response to conventional chemotherapy and to high-dose chemotherapy followed by stem cell rescue.7Moreau P Facon T Leleu X Morineau N Huyghe P Harousseau JL Bataille R Avet-Loiseau H Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy.Blood. 2002; 100: 1579-1583Crossref PubMed Scopus (278) Google Scholar The clinical significance of the t(4;14) translocation suggests that monitoring of all MM patients would enable more informed treatment decisions. Because of the cost and technical complexity of molecular diagnostics, they are not routinely used in most hospitals. Although fluorescence in situ hybridization readily detects the t(4;14) translocation,9Finelli P Fabris S Zagano S Baldini L Intini D Nobili L Lombardi L Maiolo AT Neri A Detection of t(4;14)(p16.4;q32) chromosomal translocations in multiple myeloma by double color fluorescent in situ hybridization.Blood. 1999; 94: 724-732PubMed Google Scholar fluorescence in situ hybridization in its current form is time-consuming, uses expensive probes, is labor-intensive, and requires highly skilled personnel to operate and interpret the results. The t(4;14) translocation can be identified using reverse transcriptase-polymerase chain reaction (RT-PCR) to detect hybrid IgH-MMSET transcripts from the derivative4Chesi M Nardini E Lim RS Smith KD Kuehl M Bergsagel PL The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts.Blood. 1998; 92: 3025-3034Crossref PubMed Google Scholar chromosome.1Keats JJ Maxwell CA Taylor BJ Hendzel MJ Chesi M Bergsagel PL Larratt LM Mant MJ Belch AR Pilarski LM Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.Blood. 2005; 105: 4060-4069Crossref PubMed Scopus (145) Google Scholar,3Richelda R Ronchetti D Baldini L Cro L Viggiano L Marzella R Rocchi M Otsuki T Lombardi L Maiolo AT A novel chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma involves the fibroblast growth factor receptor gene.Blood. 1997; 90: 4062-4070Crossref PubMed Google Scholar,6Winkler JM Greipp P Fonseca R t(4;14)(p16.3;q32) is strongly associated with a shorter survival in myeloma patients.Br J Haematol. 2003; 120: 170-171Crossref PubMed Scopus (11) Google Scholar,10Keats JJ Strachan E Belch AR Pilarski LM The classical illegitimate switch translocation model Is unable to account for at least half of the translocation breakpoints from t(4;14)(p16;q32) multiple myeloma (Abstract).Blood. 2005; 106: 446AGoogle Scholar,11Malgeri U Baldini L Perfetti V Fabris S Vignarelli MC Colombo G Lotti V Compasso S Bogni S Lombardi L Maiolo AT Neri A Detection of t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma by reverse transcription-polymerase chain reaction analysis of IGH-MMSET fusion transcripts.Cancer Res. 2000; 60: 4058-4061PubMed Google Scholar The integrated RT-PCR and capillary electrophoresis (CE)-based microchip detection method reported here is well suited for automated patient monitoring and could lead to routine usage at, possibly, every clinic visit. 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Ramsey and colleagues,29Khandurina J McKnight TE Jacobson SC Waters LC Foote RS Ramsey JM Integrated system for rapid PCR-based DNA analysis in microfluidic devices.Anal Chem. 2000; 72: 2995-3000Crossref PubMed Scopus (500) Google Scholar,30Waters LC Jacobson SC Kroutchinina N Khandurina J Foote RS Ramsey JM Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.Anal Chem. 1998; 70: 158-162Crossref PubMed Scopus (406) Google Scholar Mathies and collea-gues,31Woolley AT Hadley D Landre P deMello AJ Mathies RA Northrup MA Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.Anal Chem. 1996; 68: 4081-4086Crossref PubMed Scopus (701) Google Scholar,32Lagally ET Scherer JR Blazej RG Toriello NM Diep BA Ramchandani M Sensabaugh GF Riley LW Mathies RA Integrated portable genetic analysis microsystem for pathogen/infectious disease detection.Anal Chem. 2004; 76: 3162-3170Crossref PubMed Scopus (267) Google Scholar Landers and colleagues,12Ferrance JP Wu Q Giordano B Hernandez C Kwok Y Snow K Thibodeau S Landers JP Developments towards a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis.Anal Chim Acta. 2003; 500: 223-236Crossref Scopus (73) Google Scholar,33Easley CJ Karlinsey JM Landers JP On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation.Lab Chip. 2006; 6: 601-610Crossref PubMed Scopus (75) Google Scholar and other groups23Hong JW Fujii T Seki M Yamamoto T Endo I Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip.Electrophoresis. 2001; 22: 328-333Crossref PubMed Scopus (160) Google Scholar,34Koh CG Tan W Zhao MQ Ricco AJ Fan ZH Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection.Anal Chem. 2003; 75: 4591-4598Crossref PubMed Scopus (179) Google Scholar have demonstrated impressive integrations of PCR-CE systems. However, there are few reports on the integration of reverse transcription onto a microchip PCR platform16Liao C Lee G Liu H Hsieh T Luo C Miniature RT-PCR system for diagnosis of RNA-based viruses.Nucleic Acids Res. 2005; 33: e156Crossref PubMed Scopus (93) Google Scholar,28Marcus JS Anderson WF Quake SR Parallel picoliter RT-PCR assays using microfluidics.Anal Chem. 2006; 78: 956-958Crossref PubMed Scopus (160) Google Scholar,35Obeid PJ Christopoulos TK Crabtree HJ Backhouse CJ Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection.Anal Chem. 2003; 75: 288-295Crossref PubMed Scopus (223) Google Scholar and none that include chip-based CE detection. This perhaps reflects the use of a two-step RT-PCR approach that requires considerable manipulation of reagents and reaction products and increases the possibility of contamination, thereby increasing the complexity of the system and the challenges to integration of CE for product detection. Here, we have used a single-step RT-PCR approach, facilitating the integration of RT-PCR and CE on the same chip. Furthermore, testing strategies published to date do not address the need to monitor cancer at the single-cell level,36Szczepek AJ Seeberger K Wizniak J Mant MJ Belch AR Pilarski LM A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.Blood. 1998; 92: 2844-2855PubMed Google Scholar,37Anonymous Going single, but not alone (Editorial).Nat Methods. 2006; 3: 581Crossref PubMed Scopus (2) Google Scholar a critical issue in clinical management of cancer heterogeneity. In this work, microfluidic testing shows strong potential for facilitating the design of customized therapies tailored to each cancer, as well as for monitoring the response to therapy and the detection of residual cancer cells that ultimately may lead to relapse. With progress in miniaturization, these devices could become inexpensive tools for routine testing of molecular biomarkers at every clinic visit. With further development, such testing could be completed in minutes. In contrast, fluorescence in situ hybridization, the only other molecular approach in clinical practice, requires days. Our microfluidic platforms have been used for PCR thermal cycling and product analysis on-chip,38Pilarski PM Adamia S Backhouse CJ An adaptable microvalving system for on-chip polymerase chain reactions.J Immunol Methods. 2005; 305: 48-58Crossref PubMed Scopus (28) Google Scholar,39Pilarski LM Lauzon J Strachan E Adamia S Atrazhev A Belch AR Backhouse CJ Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.J Immunol Methods. 2005; 305: 94-105Crossref PubMed Scopus (17) Google Scholar detection of gene polymorphisms,40Pilarski LM Kaigala G Adamia S Chowdhury J Huskins R Lauzon J Preiksaitas J Belch AR Backhouse CJ Microfluidic devices for cancer, infectious disease and pharmacogenetics (Abstract).Transfus Med. 2005; 15: 337-367Crossref PubMed Google Scholar41Footz T Somerville MJ Tomaszewski R Elyas B Backhouse CJ Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis.Analyst. 2004; 129: 25-31Crossref PubMed Scopus (29) Google Scholar42Manage DP Zheng Y Somerville MJ Backhouse CJ On-chip HA/SSCP for the detection of hereditary haemochromatosis.Micro Nanofluidics. 2005; 1: 364-372Crossref Scopus (12) Google Scholar43Vahedi G Kaler K Backhouse CJ An integrated method for mutation detection using on-chip sample preparation, single stranded conformation polymorphism, and heteroduplex analysis.Electrophoresis. 2004; 25: 2346-2356Crossref PubMed Scopus (28) Google Scholar and detection of viral titers in unprocessed urine from renal transplant recipients18Kaigala GV Huskins RJ Preiksaitis J Pang XL Pilarski LM Backhouse CJ Automated surveillance using microfluidic chip-based PCR thermocycling and product detection to assess risk of polyoma BK virus associated neuropathy in renal transplant recipients.Electrophoresis. 2006; 27: 3753-3763Crossref PubMed Scopus (65) Google Scholar (with integrated sample preparation). Overall, on-chip PCR uses small reaction volumes,38Pilarski PM Adamia S Backhouse CJ An adaptable microvalving system for on-chip polymerase chain reactions.J Immunol Methods. 2005; 305: 48-58Crossref PubMed Scopus (28) Google Scholar with analysis of picoliters of PCR product during each microchip CE run at a sensitivity comparable with that of conventional technology requiring 10 to 200 times more sample.39Pilarski LM Lauzon J Strachan E Adamia S Atrazhev A Belch AR Backhouse CJ Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.J Immunol Methods. 2005; 305: 94-105Crossref PubMed Scopus (17) Google Scholar Here, we report on the sensitive detection of IgH MMSET hybrid transcripts and transcripts encoding clonotypic IgH VDJ gene rearrangements in bone marrow (BM) and blood cells from patients with t(4;14) MM, in aggregate populations and in individual cells. We have successfully screened patient samples for these clinically important cancer biomarkers on an RT-PCR-CE microfluidic chip, with validation against a conventional "gold standard." After Institutional Review Board approval and informed consent, blood and BM samples were obtained at diagnosis or relapse from 12 patients with multiple myeloma, including four with the MB4-1, four with the MB4-2, and four with the MB4-3 breakpoints. Blood and BM were processed as previously described.36Szczepek AJ Seeberger K Wizniak J Mant MJ Belch AR Pilarski LM A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.Blood. 1998; 92: 2844-2855PubMed Google Scholar All patients were confirmed as having the t(4;14) translocation as previously described.1Keats JJ Maxwell CA Taylor BJ Hendzel MJ Chesi M Bergsagel PL Larratt LM Mant MJ Belch AR Pilarski LM Overexpression of transcripts originating from the MMSET Locus characterizes all t(4;14)(p16;q32) positive multiple myeloma patients.Blood. 2005; 105: 4060-4069Crossref PubMed Scopus (145) Google Scholar,5Keats JJ Reiman T Maxwell CA Taylor BJ Larratt LM Mant MJ Belch AR Pilarski LM In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression.Blood. 2003; 101: 1520-1529Crossref PubMed Scopus (335) Google Scholar The chips used here are patterned poly(dimethyl)siloxane (PDMS) irreversibly bonded to a glass substrate as previously described.18Kaigala GV Huskins RJ Preiksaitis J Pang XL Pilarski LM Backhouse CJ Automated surveillance using microfluidic chip-based PCR thermocycling and product detection to assess risk of polyoma BK virus associated neuropathy in renal transplant recipients.Electrophoresis. 2006; 27: 3753-3763Crossref PubMed Scopus (65) Google Scholar For the initial phase of this study, a two-chip system was used incorporating a three-reservoir chip to perform RT-PCR and analysis using the glass CE chip. The CE chip is a two-layer glass chip from Micralyne (Edmonton, AB, Canada) as described previously.39Pilarski LM Lauzon J Strachan E Adamia S Atrazhev A Belch AR Backhouse CJ Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.J Immunol Methods. 2005; 305: 94-105Crossref PubMed Scopus (17) Google Scholar The two-chip approach increased the speed of protocol development and throughput in analysis. Where indicated, an integrated PDMS/glass chip incorporating the architecture of both of these chips (a PCR-CE chip) was used. Glass CE chips were fully reusable,44Ma R Crabtree HJ Backhouse CJ A rejuvenation method for poly(N,N-dimethlacrylamide)-coated glass microfluidic chips.Electrophoresis. 2005; 26: 2692-2700Crossref PubMed Scopus (9) Google Scholar and consistency was monitored using a standard calibration procedure.45Ma R Kaler K Backhouse CJ A rapid performance assessment method for microfluidic chips. 2004 International Conference on MEMS, Nano, and Smart Systems (ICMENS 2004), 25–27 Aug 2004, Banff, Alberta, Canada.IEEE Computer Society. 2004; 1: 680-686Google Scholar Thermal cycling was performed using a custom-designed dual-Peltier system (G. Kaigala, J. Jiang, C.J. Backhouse, and H.J. Marquez, manuscript under revision) and other physical subsystems. This chip thermal cycler is controlled using a custom-built software controller that is resident on a microcontroller of the system. Precision in control of temperature in the range of ±0.1°C at set points is achieved, and temperature ramp rates of ∼6°C/second are achieved. On-chip diaphragm micro-pumping and micro-pinch-off valving using robotic arms that takes advantage of the deforming capability of PDMS are used here as previously described,38Pilarski PM Adamia S Backhouse CJ An adaptable microvalving system for on-chip polymerase chain reactions.J Immunol Methods. 2005; 305: 48-58Crossref PubMed Scopus (28) Google Scholar thus ensuring a system that is reusable with no cross-contamination between runs. Dead volume using this micropumping approach is less than 10% of the reaction chamber volume. For the integrated PCR-CE chips, using the diaphragm pumps, fluid from the reaction chamber (after thermal cycling is completed) is dispensed into the output chamber, which is also the input well to the CE section of this integrated chip. All reaction mixes were prepared in a total volume of 25 μl for running multiple replicates of chip-based PCR and a control using the conventional thermal cycler. This 25-μl mix contained 2.5 μl of 10× PCR buffer [20 mmol/L Tris-HCl, pH 8.4, and 50 mmol/L (final concentration) KCl], 1 μl of MgCl2 (2 mmol/L), 1 μl of dNTP (0.2 mmol/L), 1.5 μl of each of the forward and reverse primers (0.2 μmol/L), 0.5 μl of Platinum Taq (0.5 U) (Invitrogen Life Technology, Burlington, ON, Canada), 2 μl of cDNA template, 2.5 μl of bovine serum albumin (1 mg/ml) (NEB, Pickering, ON, Canada) (10 mg/ml), and 13.5 μl of double distilled water. Two microliters of this mixture was introduced to each on-chip PCR chamber. This volume was chosen to ensure that sufficient template was available for the amplification reaction; smaller volumes lead to limiting dilution of templates. Peltier-based thermocycling was at 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; and a final extension of 72°C for 10 minutes. Forward and reverse primer sequences to amplify hybrid transcripts with the MB4-2 (438 bp) and MB4-3 (275 bp) breakpoints5Keats JJ Reiman T Maxwell CA Taylor BJ Larratt LM Mant MJ Belch AR Pilarski LM In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression.Blood. 2003; 101: 1520-1529Crossref PubMed Scopus (335) Google Scholar,11Malgeri U Baldini L Perfetti V Fabris S Vignarelli MC Colombo G Lotti V Compasso S Bogni S Lombardi L Maiolo AT Neri A Detection of t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma by reverse transcription-polymerase chain reaction analysis of IGH-MMSET fusion transcripts.Cancer Res. 2000; 60: 4058-4061PubMed Google Scholar were as follows: Iu1, 5′-AGCCCTTGTTAATGGACTTG-3′; and ms6r, 5′-CCTCAATTTCCCTGAAATTGGTT-3′. To detect a 343-bp product for MB4-1, the Iu1 primer was paired with the ME3 primer in MMSET exon 3: 5′-AGCTTGTCGGCTGGAATAAA-3′. The Iu1 primer was tagged with VIC (ABI, Foster City, CA). Products from representative on-chip reactions were sequenced to confirm the identity of the PCR products detected by on microchip-based CE. Individual BM plasma cells from t(4;14) myeloma were sorted into PCR tubes containing 8 μl of direct lysis buffer as previously described,36Szczepek AJ Seeberger K Wizniak J Mant MJ Belch AR Pilarski LM A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.Blood. 1998; 92: 2844-2855PubMed Google Scholar followed by reverse transcription, precipitation of the cDNA, redissolving the DNA pellet in 3 μl of PCR mixture, and transferring 2 μl to the sample reservoir of the PCR chip for 35 thermal cycles. The product was then mixed with a size standard (GS500) off-chip and transferred to a glass chip for CE in a denaturing separation matrix. The RT protocol for two-step RT-PCR involved mixing 1 μg of RNA to 1 μl of dT15 primer (10 μmol/L) and adding water to reach a 12-μl volume. Th

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