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Dried blood spot sampling for detection of monoclonal immunoglobulin gene rearrangement
Leukemia Research 37 (2013) 1265–1270
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Dried blood spot sampling for detection of monoclonal immunoglobulin gene rearrangement Maria Raffaella Petrara a , Lisa Elefanti b , Monica Quaggio a , Marisa Zanchetta b , Maria Chiara Scaini b , Nestory Masalu c , Anita De Rossi a,b , Chiara Menin b,∗ a
Department of Surgery, Oncology and Gastroenterology, Section of Immunology and Oncology, AIDS Reference Center, University of Padova, Padova, Italy Immunology and Molecular Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy c Department of Oncology, Bugando Medical Centre, P.O. Box 1370, Mwanza, Tanzania b
a r t i c l e
i n f o
Article history: Received 6 May 2013 Accepted 5 August 2013 Available online 13 August 2013 Keywords: Dried blood Clonality EBV IGH rearrangement Lymphoproliferative diseases
a b s t r a c t Molecular methods are important tools for diagnosis and monitoring of many lymphoproliferative disorders. The reliability of lymphoma diagnoses is strikingly different between developed and developing countries, partly due to lack of access to these advanced molecular analyses. To overcome these problems, we propose a new application of dried blood spots (DBS) for detecting clonal B-cell populations in peripheral blood (PB). We ensured that the DBS contained sufficient lymphocytes to perform a PCR-based clonality assay without producing false positives. Using the Namalwa B-cell line, we established that the assay is sensitive enough to detect 200 clonal cells in the analyzed sample. Very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with B-cell chronic lymphocytic leukemia. B-cell clonality can also be detected in DBS from African children with EBV-associated diseases. This is the first study demonstrating that clonality testing can be performed on DBS samples, thus improving the diagnostic and monitoring options for lymphoproliferative diseases in resource-limited settings. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction In many lymphoproliferative disorders, despite wellestablished histomorphological and immunophenotypical criteria, complementary molecular methods are required for conclusive diagnoses. According to published data, in about 10% of patients with suspected lymphoproliferative disorders, PCR-based clonality testing is useful in discriminating between reactive lymphoproliferation with polyclonally rearranged immunoglobulin or T-cell receptor genes, and malignant proliferation with clonal rearrangements [1–3]. Unfortunately, striking differences still exist in the reliability of lymphoma diagnoses between developed and developing countries. This is partly due to lack of access to these advanced molecular analyses, as occurs in several African countries in which lymphoma diagnoses are based on morphology alone. In African countries, among lymphoproliferative disorders, of particular interest are those linked to Epstein–Barr virus infection (EBV). EBV is involved in the development of a wide range of
∗ Corresponding author. Tel.: +39 0498215882; fax: +39 0498072854. E-mail address: [email protected] (C. Menin). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.08.003
B-cell malignancies, ranging from classical Hodgkin’s lymphoma (HL) to non-Hodgkin’s lymphoma (NHL), including Burkitt’s lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and immunoblastic lymphomas (IBL) in immunocompromised patients [4,5]. In Africa, primary infection with EBV occurs during infancy and early childhood. EBV-associated lymphomas are an important cause of mortality and morbidity in children, and BL in African children accounts for up to 75% of all childhood malignancies [6]. We have recently demonstrated that, in African children, NHLs are strongly associated with high levels of EBV in peripheral blood (PB) [7]. Notably, circulating malignant clones in the blood may be present in patients with late-stage lymphomas [8]. Indeed, although lymph nodes and bone marrow are the tissues of choice for detecting monoclonal populations in leukemias and lymphomas, PB has been shown to be reliable and safe in detecting malignant clones [9]. Thus, detecting clonality in the PB of patients with a high EBV load may represent a marker of tumor burden. Clonality analysis in PB requires fresh whole blood and reliable transport to laboratories with specialized equipment. As such, an easy blood collection and conservation method would improve diagnosis in most cases. One option, to simplify collaboration between institutions in developed and developing countries, and/or between small laboratories and central facilities in the
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same country, relies on the use of filter paper for blood collection; dried blood spot (DBS) sampling is widely used in many types of tests, including chemical serological and genetic applications [10–12]. DBS ensures easy sample handling, transport and storage, especially for samples collected at remote sites where the laboratory equipment, personnel, or infrastructures necessary for correct handling of blood samples may not be available. Here, we propose a new application of DBS sampling for detecting clonal B-cell populations in PB for the diagnosis and monitoring of B-cell malignancies.
and an internal TaqMan probe (5’-FAM-AAT CCT CCT ACC CTC TCT TTA TGC CAT GTG TGT-TAMRA-3’). Each PCR was performed in a 25-l reaction mix containing 5 l of sample, 12.5 l of LightCycler 480 Probes Master, 300 nM of each EBNA2 primer, and 100 nM of EBV type 1 probe. Amplification was carried out in a thermal cycler (LightCycler 480, Roche Diagnostics). A standard reference curve was obtained by five-fold serial dilution of an amplicon for EBV. The real-time PCR assay had a detection limit of 5 copies, with a dynamic range from 5 to 2 × 105 copies.
2. Materials and methods
One of the limitations of DBS for laboratory diagnosis is the small quantity of sample. Since single-cell DNA can be amplified by generating a single (clonal) amplification product, a false positive result may occur if there are very few normal lymphocytes in the test sample. To rule out this possibility, although the BIOMED-2 protocol indicates that at least 100 ng of DNA should be used for each PCR reaction, we first verified whether 50 ng of DNA from normal whole blood (corresponding to about 7500 cells) was sufficient to detect a normal polyclonal pattern corresponding to B-lymphocytes carrying the different immunoglobulin gene rearrangements. DNA was extracted from 500 l of whole blood from five donors, and 50 ng of each sample were analyzed for FR1-JH, FR2-JH, FR3-JH rearrangements of the IGH gene; a polyclonal pattern was observed in all cases. From these same donors, 50 l of blood were spotted on DBS filters and 5 l (about 50 ng) of the 50 l DNA extracted were analyzed; polyclonal results were concordant with those obtained with DNA from fresh blood (Fig. 1). Thus, 5 l of spotted blood contain sufficient lymphocytes to perform an IGH clonality assay without false positives.
2.1. Blood, cell line and DBS samples Blood was taken from five consenting healthy volunteer donors at the Instituto Oncologico Veneto, IOV-IRCCS, of Padova, Italy. The samples were tested to confirm lack of EBV-DNA. Six B-cell chronic lymphocytic leukemia (B-CLL) samples were referred to the IOV-IRCCS diagnostic laboratory for routine clonality testing, to complete the pathologist’s diagnosis of B-CLL. All venous blood samples were collected in EDTA vacutainer tubes, and parallel samples were prepared: 50 l of blood were spotted on Protein Saver TM 903 Cards (Whatman GmbH, Hahnestra, Germany), dried overnight at room temperature; 500 l of whole blood were transferred to Eppendorf tubes for DNA extraction. The Namalwa cell line, an EBV-positive BL line which is known to carry two integrated EBV type 1 genomes per cell [13], was established at the initial concentration of 106 cells/ml and diluted into whole blood from healthy donors to obtain 200–150–100–50–40–20–2–0 Namalwa cells per l of blood. Fifty l of each dilution was spotted on filter paper cards. All DBS samples were prepared in duplicate. A pellet with Namalwa cells only was used as a positive control of monoclonality. DBS samples from African children with NHL and age-matched controls were collected at the Bugando Medical Centre in Tanzania; DBS cards were stored at room temperature in individual ziplock bags containing a desiccant, and sent to the IOVIRCCS of Padova, Italy [7]. In this study, we analyzed 13 available samples. 2.2. DNA extraction Genomic DNA was isolated with the automated MagNA Pure Compact instrument (Roche Applied Science, Indianapolis, IN). Briefly, 500 l of fresh blood were processed according to the manufacturer’s protocol contained in the MagNA Pure Compact Nucleic Acid Isolation kit I-Large Volume (Roche Applied Science), with elution volume set at 200 l. For DNA extraction from DBS cards, one spot with 50 l of dried blood was cut into pieces manually, avoiding cross-contamination. An external lysis step was performed in 180 l of MagNA pure DNA tissue lysis buffer (Roche Applied Science) and 20 l of proteinase K solutions at 56 ◦ C overnight. After inactivation of proteinase K at 90 ◦ C for 10 min and adjustment of the final volume to 500 l with added phosphate-buffered saline (PBS), the DNA was extracted with the above protocol, with elution volume set at 50 l, which is the minimum permitted by the protocol. The concentration of DNA extracted was determined on a spectrophotometer (NanoDrop ND-1000, Wilmington, DE). Approximately 11–24 ng/l of DNA were recovered from the DBS cards. 2.3. Clonality assay The extracted DNA samples were tested for B-cell immunoglobulin heavy chain (IGH) clonality with the IGH gene clonality assay (Invivoscribe Technologies, San Diego, CA), containing BIOMED-2 primers, positive and negative controls, in accordance with the manufacturer’s instructions. As in specimens with limited DNA the recommended multiplex PCRs for suspected B-cell proliferation are the three framework subregions (FR) of the IGH gene, preferably followed by immunoglobulin kappa targets [14], the IGH FR1, FR2 and FR3 Master Mix was used, as well as the Specimen Control Size Ladder Master Mix for template amplification control. The combined use of a three FR multiplex strategy significantly improves clonality detection in mature B-cell malignancies, and the presence of clonal rearrangements can be detected in 89% of all B-cell diseases [15]. Each PCR reaction was prepared with the maximum volume of DNA provided in the protocol (5 l) for DBS cards or 50 ng of DNA extracted directly from fresh blood. These post-PCR products were visualized by capillary electrophoresis on an ABI Prism 3730XL Genetic Analyzer, with subsequent analysis by ABI GeneMapper 4.0 software (Applied Biosystems). All samples were tested twice. A sample was considered to be clonal only when one or two reproducible peaks were detected within the valid range. Known non-specific peaks were excluded, in order to avoid false positives [1]. 2.4. EBV-DNA quantification EBV-DNA levels were quantified by real-time PCR assay, as previously described [7,16]. Briefly, a 106 base-pair EBNA2 gene fragment is amplified with a primer pair (Fw 5 -CTG CCC ACC CTG AGG ATT TCC-3 and Rv 5 -CTG CCA CCT GGC GGC AAC-3 )
3. Results 3.1. Evaluation of polyclonal B cells on DBS
3.2. Lower limit for detecting monoclonal IGH gene rearrangement on DBS We assessed the lower limit of clonal detection in the PCR-based analysis of DBS samples with Namalwa B cells, diluted in the whole blood of a healthy donor; 50 l of each dilution were spotted on DBS filters in duplicate. As Namalwa cells contain two integrated EBV genomes/cell [13], the number of Namalwa B clonal cells contained in samples diluted from DBS and analyzed for clonality was also verified by testing them for EBV-DNA copy number. The number of EBV copies was estimated by real-time PCR and the values were closely related to those expected (r = 0.987, p < 0.0001). DNA from 100% Namalwa cells (N) revealed a monoclonal IGH gene rearrangement in all three framework regions. With DNA from Namalwa cells diluted in normal blood, the highest sensitive clonal signal was obtained with primers amplifying sequences between the FR2-JH and FR3-JH regions. As shown in Fig. 2, a positive clonal signal could be detected in DBS samples containing 40 Namalwa cells per l of blood. DBS samples with 20 or 2 Namalwa cells/l revealed only a polyclonal pattern, similar to DBS spotted only with normal PB. Thus, clonal B-cell populations on DBS can be detected by PCRbased methods when represented by at least 200 clonal cells in the sample. 3.3. Clonality analysis on DBS from B-CLL and EBV-NHL We tested the reliability of our approach for detecting clonality on DBS from patients with B-CLL. Equal amounts of DNA, either extracted from DBS or from fresh blood samples taken from 6 BCLL patients, were analyzed by the same PCR-based approach. As shown in Fig. 3A, very similar profiles, with similar migration distances and heights of clonal peaks, were present in both DBS and fresh blood samples from each patient. We also analyzed DBS from 13 EBV-positive African children (ranging from 2525 to 35,506 EBV copies/ml of blood): 4 NHL and 9 controls. All samples were
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Fig. 1. IGH gene polyclonality analysis. (A) IGH FR1-JH, (B) FR2-JH and (C) FR3-JH multiplex PCR GeneScan analysis with polyclonal controls (upper panels), 50 ng of DNA extracted from DBS (middle panels) and fresh blood (lower panels) from one representative healthy donor, showing typical polyclonal Gaussian patterns.
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Fig. 2. Minimal detection of B-cell clonality. Namalwa B-cells (N) were serially diluted in normal blood (panels with 200, 150, 100, 50, 40, 20, 2 N/l of blood). DBS cards were prepared with 50 l of each dilution, and 5 l of 50 l DNA extracted were analyzed by multiplex PCR for IGH gene rearrangements. From a representative case, GeneScanning revealed a distinct monoclonal peak with expected position of 100% N, still detected at dilution of 40 N/l by PCR with Master Mix specific for (A) FR2-JH and (B) FR3-JH rearrangements.
analyzed in duplicate and proved suitable for clonality testing. None of the 9 controls disclosed a clonal result, thus supporting the lack of false positive results, while a monoclonal IGH rearrangement was found in NHL sample #4 (Fig. 3B). Notably, NHL #1, #2, and #3 were in stage I, whereas NHL #4 was in stage III and had the highest EBV level; this result matches the finding that malignant clones are more easily detected in peripheral blood in the late stage of the disease [8]. 4. Discussion DBS cards turn out to be feasible for diagnosing and monitoring various diseases in the developing world. We assessed whether DBS sampling was also suitable to verify the presence of B-cell clonal populations circulating in the blood by immunoglobulin gene rearrangement analysis, with a view to offering molecular evaluation to the diagnosis and monitoring of lymphoproliferative diseases in resource-limited settings. The first problem is whether the DBS contains a sufficient number of B lymphocytes to detect normal polyclonal B-cell populations by BIOMED-2 protocol PCR analysis. This is an important aspect,
in order to avoid possible false positives (pseudoclonality) due to amplification of the few immunoglobulin gene rearrangements deriving from a limited number of B-cells in the sample analyzed. Although the BIOMED-2 guidelines suggest using at least 100 ng of DNA, our experiments showed that about 50 ng of extracted DNA, corresponding to that from 5 l of the spotted blood (corresponding to approximately 750–3600 B cells) sufficed to yield the expected polyclonal pattern in each specific PCR reaction, even when normal fresh blood was analyzed. Notably, while a previous study reported that 20 ng of DNA from normal tonsil, corresponding to 6000–9500 B cells, is required to detect a complete polyclonal pattern [17], we found that, with the multiplex PCR method and fluorescent analysis, less than half this number of B-cells was sufficient. The second problem to be evaluated was the sensitivity of the DBS approach in detecting clonal cell populations circulating in the blood. Since limited sensitivity may hamper the detection of small clonal cell populations, we determined how many B clonal cells (Namalwa) must be present in the blood for detection, with DNA corresponding to 5 l of spotted blood. We demonstrated that the monoclonal pattern could be identified even in the sample with 200 clonal Namalwa cells. Since a normal adult lymphocyte
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Fig. 3. IGH gene clonality analysis in B-CLL and EBV-NHL. (A) Three representative B-CLL cases (B-CLL #1, B-CLL #2, B-CLL #3) were analyzed with fresh whole blood in parallel with DBS cards with 50 l of blotted blood. (B) DBS from 4 African children with diagnosis of EBV-NHL (NHL #4, NHL #1, NHL #2, NHL #3) were analyzed by clonality assay. NHL #4 disclosed monoclonal pattern characterized by two predominant peaks over polyclonal background. In all cases, GeneScan analysis of multiplex PCR for FR1-JH rearrangements revealed monoclonal peak with DNA from fresh blood and DBS cards.
count is usually between 1000 and 4800 lymphocytes/l of blood, of which approximately 15% are B-cells (about 400 B polyclonal lymphocytes/l of blood) [18], the detection of 40 clonal B-cells per l of blood corresponds to a sensitivity of 10% with the BIOMED2 PCR protocol with DBS cards. Since the detection limit depends not only on the technique applied, but also on the relative size of the ‘background’ of normal (polyclonal) B-lymphocytes, it is not surprising that the sensitivity is lower with DBS cards than with fresh blood samples, in which the BIOMED-2 assay can reliably detect even 1–5 positive cells per 100 normal lymphocytes (1–5% of sensitivity) [1]. We also demonstrated that the same clonal results could be obtained by testing fresh blood samples and DBS cards from the same patients. We used blood from B-CLL cases to make this comparison, because this malignancy is characterized by a high number of clonal tumor cells in peripheral blood: clinical diagnosis of BCLL is in fact defined by absolute lymphocytosis of at least 5000/l cells with an appropriate immunophenotype [19]. In all cases analyzed, we found the same clonal profile, using DNA from both DBS compared with fresh blood. A diagnosis of leukemia is not usually problematic: immnophenotyping and cytogenetic analysis and histochemical staining can establish diagnosis in virtually all leukemia cases. However, a diagnosis of leukemia may sometimes be more complicated, and molecular clonality evaluation is thought to produce
valuable additional information. DBS cards may be useful tools when the molecular analyses complementary to histomorphological/immunophenotypic evaluation are not available. Since the presence of clonal lymphomatous cells in the blood is well recognized in most patients with B-lymphoma [8,9,20–24], blood cell samples may represent another easily accessible biomaterial to monitor these patients. Notably, in view of the low frequency of circulating clonal B cells in some cases, a negative DBS clonality test result is unlikely to rule out a diagnosis of lymphoma. However, a positive result may predict tumor burden and allow greater monitoring of patients during treatment. In addition, since clonality does not always imply malignancy, because some reactive processes contain large clonal lymphocyte populations, clonal results should always be used in the clinical context. We thought this method would be particularly useful in developing countries, for patients at high risk of lymphoma and in whom more invasive sampling is difficult. Since in Africa there is a striking association of EBV load with pediatric lymphomas [7], DBS sampling would be useful to test clonality in children with high EBV load in PB. We tested some DBS samples from Tanzanian children; all controls and 3 NHL cases at early stage I disclosed polyclonal patterns of B-cells, but the Bcell clonal population was present in the DBS from one patient with EBV-NHL at stage III, indicating a high tumor burden. This is the first study showing that clonality testing can be performed by DBS sampling, suggesting a new potential strategy for
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improving diagnosis and monitoring of EBV-related and unrelated lymphoproliferative diseases in resource-limited settings. Conflict of interest statement All authors have no conflict of interest to report. Acknowledgments This study was partly funded by grants from the Italian Association for Cancer Research (AIRC) and the “Programma Integrato Oncologia (RO4/2007)”. The authors would like to thank Emma D’Andrea and Luigi Chieco-Bianchi for their critical reading of the manuscript. They also thank Rogatus Kabyemera of the Department of Pediatrics and Child Health, Bugando Medical Centre, Mwanza, Tanzania, for African DBS samples. Contributions: C.M. and A.D.R. provided the conception and design of the study, drifting the article, revised it critically for important intellectual content, and final approval of the version to be submitted; M.R.P. performed the research, analyzed the data and drafting of manuscript; L.E. and M.Q. performed all experiments; M.Z. and M.C.S. contributed to the technique support; N.M. collected African samples and provided clinical annotations. All authors have read and approved the final version of the manuscript. References [1] van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT983936. Leukemia 2003;17:2257–317. [2] van Dongen JJ, Wolvers-Tettero IL. Analysis of immunoglobulin and T cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clin Chim Acta 1991;198:93–174. [3] van Krieken JH, Langerak AW, Macintyre EA, Kneba M, Hodges E, Sanz RG, et al. Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 concerted action BHM4-CT98-3936. Leukemia 2007;21:201–6. [4] Carbone A, Gloghini A, Dotti G. EBV-associated lymphoproliferative disorders: classification and treatment. Oncologist 2008;13:577–85. [5] Ometto L, Menin C, Masiero S, Bonaldi L, Del Mistro A, Cattelan AM, et al. Molecular profile of Epstein–Barr virus in human immunodeficiency virus type 1-related lymphadenopathies and lymphomas. Blood 1997;90:313–22. [6] Cader FZ, Kearns P, Young L, Murray P, Vockerodt M. The contribution of the Epstein–Barr virus to the pathogenesis of childhood lymphomas. Cancer Treat Rev 2010;36:348–53.
[7] Kabyemera R, Masalu N, Rambau P, Kamugisha E, Kidenya B, De Rossi A, et al. Relationship between non-Hodgkin’s lymphoma and blood levels of Epstein–Barr virus in children in north-western Tanzania: a case control study. BMC Pediatr 2013;13:4. [8] Muringampurath-John D, Jaye DL, Flowers CR, Saxe D, Chen Z, Lechowicz MJ, et al. Characteristics and outcomes of diffuse large B-cell lymphoma presenting in leukaemic phase. Br J Haematol 2012;158:608–14. [9] Leal E, Esparza-Flores MA, Lopez-Guido B, Aguilar-Luna C, Aguilar-Lopez L, Jaloma-Cruz AR, et al. Detection and monitoring of clonality in peripheral blood and bone marrow of patients with B-cell lymphoproliferative disorders. Hematol Oncol 2003;21:25–31. [10] McDade TW, Williams S, Snodgrass JJ. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 2007;44:899–925. [11] Mei JV, Alexander JR, Adam BW, Hannon WH. Use of filter paper for the collection and analysis of human whole blood specimens. J Nutr 2001;131: 1631S–6S. [12] Snijdewind IJ, van Kampen JJ, Fraaij PL, van der Ende ME, Osterhaus AD, Gruters RA. Current and future applications of dried blood spots in viral disease management. Antiviral Res 2012;93:309–21. [13] Lawrence JB, Villnave CA, Singer RH. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 1988;52:51–61. [14] Langerak AW, Groenen PJ, Bruggemann M, Beldjord K, Bellan C, Bonello L, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 2012;26:2159–71. [15] Evans PA, Pott C, Groenen PJ, Salles G, Davi F, Berger F, et al. Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 2007;21:207–14. [16] Petrara MR, Cattelan AM, Zanchetta M, Sasset L, Freguja R, Gianesin K, et al. Epstein–Barr virus load and immune activation in human immunodeficiency virus type 1-infected patients. J Clin Virol 2012;53:195– 200. [17] Hoeve MA, Krol AD, Philippo K, Derksen PW, Veenendaal RA, Schuuring E, et al. Limitations of clonality analysis of B cell proliferations using CDR3 polymerase chain reaction. Mol Pathol 2000;53:194–200. [18] Ollila J, Vihinen M. B cells. Int J Biochem Cell Biol 2005;37:518–23. [19] Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:4990–7. [20] Berinstein NL, Reis MD, Ngan BY, Sawka CA, Jamal HH, Kuzniar B. Detection of occult lymphoma in the peripheral blood and bone marrow of patients with untreated early-stage and advanced-stage follicular lymphoma. J Clin Oncol 1993;11:1344–52. [21] Frickhofen N, Muller E, Sandherr M, Binder T, Bangerter M, Wiest C, et al. Rearranged Ig heavy chain DNA is detectable in cell-free blood samples of patients with B-cell neoplasia. Blood 1997;90:4953–60. [22] Horning SJ, Galili N, Cleary M, Sklar J. Detection of non-Hodgkin’s lymphoma in the peripheral blood by analysis of antigen receptor gene rearrangements: results of a prospective study. Blood 1990;75:1139–45. [23] Jones RJ, Gocke CD, Kasamon YL, Miller CB, Perkins B, Barber JP, et al. Circulating clonotypic B cells in classic Hodgkin lymphoma. Blood 2009;113:5920–6. [24] Yeh CH, Tseng R, Albitar M. Plasma-based detection of clonality in lymphoid malignancies. Eur J Haematol 2009;82:450–3.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Dried blood spot sampling for detection of monoclonal immunoglobulin gene rearrangement Maria Raffaella Petrara a , Lisa Elefanti b , Monica Quaggio a , Marisa Zanchetta b , Maria Chiara Scaini b , Nestory Masalu c , Anita De Rossi a,b , Chiara Menin b,∗ a
Department of Surgery, Oncology and Gastroenterology, Section of Immunology and Oncology, AIDS Reference Center, University of Padova, Padova, Italy Immunology and Molecular Oncology, Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy c Department of Oncology, Bugando Medical Centre, P.O. Box 1370, Mwanza, Tanzania b
a r t i c l e
i n f o
Article history: Received 6 May 2013 Accepted 5 August 2013 Available online 13 August 2013 Keywords: Dried blood Clonality EBV IGH rearrangement Lymphoproliferative diseases
a b s t r a c t Molecular methods are important tools for diagnosis and monitoring of many lymphoproliferative disorders. The reliability of lymphoma diagnoses is strikingly different between developed and developing countries, partly due to lack of access to these advanced molecular analyses. To overcome these problems, we propose a new application of dried blood spots (DBS) for detecting clonal B-cell populations in peripheral blood (PB). We ensured that the DBS contained sufficient lymphocytes to perform a PCR-based clonality assay without producing false positives. Using the Namalwa B-cell line, we established that the assay is sensitive enough to detect 200 clonal cells in the analyzed sample. Very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with B-cell chronic lymphocytic leukemia. B-cell clonality can also be detected in DBS from African children with EBV-associated diseases. This is the first study demonstrating that clonality testing can be performed on DBS samples, thus improving the diagnostic and monitoring options for lymphoproliferative diseases in resource-limited settings. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction In many lymphoproliferative disorders, despite wellestablished histomorphological and immunophenotypical criteria, complementary molecular methods are required for conclusive diagnoses. According to published data, in about 10% of patients with suspected lymphoproliferative disorders, PCR-based clonality testing is useful in discriminating between reactive lymphoproliferation with polyclonally rearranged immunoglobulin or T-cell receptor genes, and malignant proliferation with clonal rearrangements [1–3]. Unfortunately, striking differences still exist in the reliability of lymphoma diagnoses between developed and developing countries. This is partly due to lack of access to these advanced molecular analyses, as occurs in several African countries in which lymphoma diagnoses are based on morphology alone. In African countries, among lymphoproliferative disorders, of particular interest are those linked to Epstein–Barr virus infection (EBV). EBV is involved in the development of a wide range of
∗ Corresponding author. Tel.: +39 0498215882; fax: +39 0498072854. E-mail address: [email protected] (C. Menin). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.08.003
B-cell malignancies, ranging from classical Hodgkin’s lymphoma (HL) to non-Hodgkin’s lymphoma (NHL), including Burkitt’s lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and immunoblastic lymphomas (IBL) in immunocompromised patients [4,5]. In Africa, primary infection with EBV occurs during infancy and early childhood. EBV-associated lymphomas are an important cause of mortality and morbidity in children, and BL in African children accounts for up to 75% of all childhood malignancies [6]. We have recently demonstrated that, in African children, NHLs are strongly associated with high levels of EBV in peripheral blood (PB) [7]. Notably, circulating malignant clones in the blood may be present in patients with late-stage lymphomas [8]. Indeed, although lymph nodes and bone marrow are the tissues of choice for detecting monoclonal populations in leukemias and lymphomas, PB has been shown to be reliable and safe in detecting malignant clones [9]. Thus, detecting clonality in the PB of patients with a high EBV load may represent a marker of tumor burden. Clonality analysis in PB requires fresh whole blood and reliable transport to laboratories with specialized equipment. As such, an easy blood collection and conservation method would improve diagnosis in most cases. One option, to simplify collaboration between institutions in developed and developing countries, and/or between small laboratories and central facilities in the
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same country, relies on the use of filter paper for blood collection; dried blood spot (DBS) sampling is widely used in many types of tests, including chemical serological and genetic applications [10–12]. DBS ensures easy sample handling, transport and storage, especially for samples collected at remote sites where the laboratory equipment, personnel, or infrastructures necessary for correct handling of blood samples may not be available. Here, we propose a new application of DBS sampling for detecting clonal B-cell populations in PB for the diagnosis and monitoring of B-cell malignancies.
and an internal TaqMan probe (5’-FAM-AAT CCT CCT ACC CTC TCT TTA TGC CAT GTG TGT-TAMRA-3’). Each PCR was performed in a 25-l reaction mix containing 5 l of sample, 12.5 l of LightCycler 480 Probes Master, 300 nM of each EBNA2 primer, and 100 nM of EBV type 1 probe. Amplification was carried out in a thermal cycler (LightCycler 480, Roche Diagnostics). A standard reference curve was obtained by five-fold serial dilution of an amplicon for EBV. The real-time PCR assay had a detection limit of 5 copies, with a dynamic range from 5 to 2 × 105 copies.
2. Materials and methods
One of the limitations of DBS for laboratory diagnosis is the small quantity of sample. Since single-cell DNA can be amplified by generating a single (clonal) amplification product, a false positive result may occur if there are very few normal lymphocytes in the test sample. To rule out this possibility, although the BIOMED-2 protocol indicates that at least 100 ng of DNA should be used for each PCR reaction, we first verified whether 50 ng of DNA from normal whole blood (corresponding to about 7500 cells) was sufficient to detect a normal polyclonal pattern corresponding to B-lymphocytes carrying the different immunoglobulin gene rearrangements. DNA was extracted from 500 l of whole blood from five donors, and 50 ng of each sample were analyzed for FR1-JH, FR2-JH, FR3-JH rearrangements of the IGH gene; a polyclonal pattern was observed in all cases. From these same donors, 50 l of blood were spotted on DBS filters and 5 l (about 50 ng) of the 50 l DNA extracted were analyzed; polyclonal results were concordant with those obtained with DNA from fresh blood (Fig. 1). Thus, 5 l of spotted blood contain sufficient lymphocytes to perform an IGH clonality assay without false positives.
2.1. Blood, cell line and DBS samples Blood was taken from five consenting healthy volunteer donors at the Instituto Oncologico Veneto, IOV-IRCCS, of Padova, Italy. The samples were tested to confirm lack of EBV-DNA. Six B-cell chronic lymphocytic leukemia (B-CLL) samples were referred to the IOV-IRCCS diagnostic laboratory for routine clonality testing, to complete the pathologist’s diagnosis of B-CLL. All venous blood samples were collected in EDTA vacutainer tubes, and parallel samples were prepared: 50 l of blood were spotted on Protein Saver TM 903 Cards (Whatman GmbH, Hahnestra, Germany), dried overnight at room temperature; 500 l of whole blood were transferred to Eppendorf tubes for DNA extraction. The Namalwa cell line, an EBV-positive BL line which is known to carry two integrated EBV type 1 genomes per cell [13], was established at the initial concentration of 106 cells/ml and diluted into whole blood from healthy donors to obtain 200–150–100–50–40–20–2–0 Namalwa cells per l of blood. Fifty l of each dilution was spotted on filter paper cards. All DBS samples were prepared in duplicate. A pellet with Namalwa cells only was used as a positive control of monoclonality. DBS samples from African children with NHL and age-matched controls were collected at the Bugando Medical Centre in Tanzania; DBS cards were stored at room temperature in individual ziplock bags containing a desiccant, and sent to the IOVIRCCS of Padova, Italy [7]. In this study, we analyzed 13 available samples. 2.2. DNA extraction Genomic DNA was isolated with the automated MagNA Pure Compact instrument (Roche Applied Science, Indianapolis, IN). Briefly, 500 l of fresh blood were processed according to the manufacturer’s protocol contained in the MagNA Pure Compact Nucleic Acid Isolation kit I-Large Volume (Roche Applied Science), with elution volume set at 200 l. For DNA extraction from DBS cards, one spot with 50 l of dried blood was cut into pieces manually, avoiding cross-contamination. An external lysis step was performed in 180 l of MagNA pure DNA tissue lysis buffer (Roche Applied Science) and 20 l of proteinase K solutions at 56 ◦ C overnight. After inactivation of proteinase K at 90 ◦ C for 10 min and adjustment of the final volume to 500 l with added phosphate-buffered saline (PBS), the DNA was extracted with the above protocol, with elution volume set at 50 l, which is the minimum permitted by the protocol. The concentration of DNA extracted was determined on a spectrophotometer (NanoDrop ND-1000, Wilmington, DE). Approximately 11–24 ng/l of DNA were recovered from the DBS cards. 2.3. Clonality assay The extracted DNA samples were tested for B-cell immunoglobulin heavy chain (IGH) clonality with the IGH gene clonality assay (Invivoscribe Technologies, San Diego, CA), containing BIOMED-2 primers, positive and negative controls, in accordance with the manufacturer’s instructions. As in specimens with limited DNA the recommended multiplex PCRs for suspected B-cell proliferation are the three framework subregions (FR) of the IGH gene, preferably followed by immunoglobulin kappa targets [14], the IGH FR1, FR2 and FR3 Master Mix was used, as well as the Specimen Control Size Ladder Master Mix for template amplification control. The combined use of a three FR multiplex strategy significantly improves clonality detection in mature B-cell malignancies, and the presence of clonal rearrangements can be detected in 89% of all B-cell diseases [15]. Each PCR reaction was prepared with the maximum volume of DNA provided in the protocol (5 l) for DBS cards or 50 ng of DNA extracted directly from fresh blood. These post-PCR products were visualized by capillary electrophoresis on an ABI Prism 3730XL Genetic Analyzer, with subsequent analysis by ABI GeneMapper 4.0 software (Applied Biosystems). All samples were tested twice. A sample was considered to be clonal only when one or two reproducible peaks were detected within the valid range. Known non-specific peaks were excluded, in order to avoid false positives [1]. 2.4. EBV-DNA quantification EBV-DNA levels were quantified by real-time PCR assay, as previously described [7,16]. Briefly, a 106 base-pair EBNA2 gene fragment is amplified with a primer pair (Fw 5 -CTG CCC ACC CTG AGG ATT TCC-3 and Rv 5 -CTG CCA CCT GGC GGC AAC-3 )
3. Results 3.1. Evaluation of polyclonal B cells on DBS
3.2. Lower limit for detecting monoclonal IGH gene rearrangement on DBS We assessed the lower limit of clonal detection in the PCR-based analysis of DBS samples with Namalwa B cells, diluted in the whole blood of a healthy donor; 50 l of each dilution were spotted on DBS filters in duplicate. As Namalwa cells contain two integrated EBV genomes/cell [13], the number of Namalwa B clonal cells contained in samples diluted from DBS and analyzed for clonality was also verified by testing them for EBV-DNA copy number. The number of EBV copies was estimated by real-time PCR and the values were closely related to those expected (r = 0.987, p < 0.0001). DNA from 100% Namalwa cells (N) revealed a monoclonal IGH gene rearrangement in all three framework regions. With DNA from Namalwa cells diluted in normal blood, the highest sensitive clonal signal was obtained with primers amplifying sequences between the FR2-JH and FR3-JH regions. As shown in Fig. 2, a positive clonal signal could be detected in DBS samples containing 40 Namalwa cells per l of blood. DBS samples with 20 or 2 Namalwa cells/l revealed only a polyclonal pattern, similar to DBS spotted only with normal PB. Thus, clonal B-cell populations on DBS can be detected by PCRbased methods when represented by at least 200 clonal cells in the sample. 3.3. Clonality analysis on DBS from B-CLL and EBV-NHL We tested the reliability of our approach for detecting clonality on DBS from patients with B-CLL. Equal amounts of DNA, either extracted from DBS or from fresh blood samples taken from 6 BCLL patients, were analyzed by the same PCR-based approach. As shown in Fig. 3A, very similar profiles, with similar migration distances and heights of clonal peaks, were present in both DBS and fresh blood samples from each patient. We also analyzed DBS from 13 EBV-positive African children (ranging from 2525 to 35,506 EBV copies/ml of blood): 4 NHL and 9 controls. All samples were
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Fig. 1. IGH gene polyclonality analysis. (A) IGH FR1-JH, (B) FR2-JH and (C) FR3-JH multiplex PCR GeneScan analysis with polyclonal controls (upper panels), 50 ng of DNA extracted from DBS (middle panels) and fresh blood (lower panels) from one representative healthy donor, showing typical polyclonal Gaussian patterns.
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Fig. 2. Minimal detection of B-cell clonality. Namalwa B-cells (N) were serially diluted in normal blood (panels with 200, 150, 100, 50, 40, 20, 2 N/l of blood). DBS cards were prepared with 50 l of each dilution, and 5 l of 50 l DNA extracted were analyzed by multiplex PCR for IGH gene rearrangements. From a representative case, GeneScanning revealed a distinct monoclonal peak with expected position of 100% N, still detected at dilution of 40 N/l by PCR with Master Mix specific for (A) FR2-JH and (B) FR3-JH rearrangements.
analyzed in duplicate and proved suitable for clonality testing. None of the 9 controls disclosed a clonal result, thus supporting the lack of false positive results, while a monoclonal IGH rearrangement was found in NHL sample #4 (Fig. 3B). Notably, NHL #1, #2, and #3 were in stage I, whereas NHL #4 was in stage III and had the highest EBV level; this result matches the finding that malignant clones are more easily detected in peripheral blood in the late stage of the disease [8]. 4. Discussion DBS cards turn out to be feasible for diagnosing and monitoring various diseases in the developing world. We assessed whether DBS sampling was also suitable to verify the presence of B-cell clonal populations circulating in the blood by immunoglobulin gene rearrangement analysis, with a view to offering molecular evaluation to the diagnosis and monitoring of lymphoproliferative diseases in resource-limited settings. The first problem is whether the DBS contains a sufficient number of B lymphocytes to detect normal polyclonal B-cell populations by BIOMED-2 protocol PCR analysis. This is an important aspect,
in order to avoid possible false positives (pseudoclonality) due to amplification of the few immunoglobulin gene rearrangements deriving from a limited number of B-cells in the sample analyzed. Although the BIOMED-2 guidelines suggest using at least 100 ng of DNA, our experiments showed that about 50 ng of extracted DNA, corresponding to that from 5 l of the spotted blood (corresponding to approximately 750–3600 B cells) sufficed to yield the expected polyclonal pattern in each specific PCR reaction, even when normal fresh blood was analyzed. Notably, while a previous study reported that 20 ng of DNA from normal tonsil, corresponding to 6000–9500 B cells, is required to detect a complete polyclonal pattern [17], we found that, with the multiplex PCR method and fluorescent analysis, less than half this number of B-cells was sufficient. The second problem to be evaluated was the sensitivity of the DBS approach in detecting clonal cell populations circulating in the blood. Since limited sensitivity may hamper the detection of small clonal cell populations, we determined how many B clonal cells (Namalwa) must be present in the blood for detection, with DNA corresponding to 5 l of spotted blood. We demonstrated that the monoclonal pattern could be identified even in the sample with 200 clonal Namalwa cells. Since a normal adult lymphocyte
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Fig. 3. IGH gene clonality analysis in B-CLL and EBV-NHL. (A) Three representative B-CLL cases (B-CLL #1, B-CLL #2, B-CLL #3) were analyzed with fresh whole blood in parallel with DBS cards with 50 l of blotted blood. (B) DBS from 4 African children with diagnosis of EBV-NHL (NHL #4, NHL #1, NHL #2, NHL #3) were analyzed by clonality assay. NHL #4 disclosed monoclonal pattern characterized by two predominant peaks over polyclonal background. In all cases, GeneScan analysis of multiplex PCR for FR1-JH rearrangements revealed monoclonal peak with DNA from fresh blood and DBS cards.
count is usually between 1000 and 4800 lymphocytes/l of blood, of which approximately 15% are B-cells (about 400 B polyclonal lymphocytes/l of blood) [18], the detection of 40 clonal B-cells per l of blood corresponds to a sensitivity of 10% with the BIOMED2 PCR protocol with DBS cards. Since the detection limit depends not only on the technique applied, but also on the relative size of the ‘background’ of normal (polyclonal) B-lymphocytes, it is not surprising that the sensitivity is lower with DBS cards than with fresh blood samples, in which the BIOMED-2 assay can reliably detect even 1–5 positive cells per 100 normal lymphocytes (1–5% of sensitivity) [1]. We also demonstrated that the same clonal results could be obtained by testing fresh blood samples and DBS cards from the same patients. We used blood from B-CLL cases to make this comparison, because this malignancy is characterized by a high number of clonal tumor cells in peripheral blood: clinical diagnosis of BCLL is in fact defined by absolute lymphocytosis of at least 5000/l cells with an appropriate immunophenotype [19]. In all cases analyzed, we found the same clonal profile, using DNA from both DBS compared with fresh blood. A diagnosis of leukemia is not usually problematic: immnophenotyping and cytogenetic analysis and histochemical staining can establish diagnosis in virtually all leukemia cases. However, a diagnosis of leukemia may sometimes be more complicated, and molecular clonality evaluation is thought to produce
valuable additional information. DBS cards may be useful tools when the molecular analyses complementary to histomorphological/immunophenotypic evaluation are not available. Since the presence of clonal lymphomatous cells in the blood is well recognized in most patients with B-lymphoma [8,9,20–24], blood cell samples may represent another easily accessible biomaterial to monitor these patients. Notably, in view of the low frequency of circulating clonal B cells in some cases, a negative DBS clonality test result is unlikely to rule out a diagnosis of lymphoma. However, a positive result may predict tumor burden and allow greater monitoring of patients during treatment. In addition, since clonality does not always imply malignancy, because some reactive processes contain large clonal lymphocyte populations, clonal results should always be used in the clinical context. We thought this method would be particularly useful in developing countries, for patients at high risk of lymphoma and in whom more invasive sampling is difficult. Since in Africa there is a striking association of EBV load with pediatric lymphomas [7], DBS sampling would be useful to test clonality in children with high EBV load in PB. We tested some DBS samples from Tanzanian children; all controls and 3 NHL cases at early stage I disclosed polyclonal patterns of B-cells, but the Bcell clonal population was present in the DBS from one patient with EBV-NHL at stage III, indicating a high tumor burden. This is the first study showing that clonality testing can be performed by DBS sampling, suggesting a new potential strategy for
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improving diagnosis and monitoring of EBV-related and unrelated lymphoproliferative diseases in resource-limited settings. Conflict of interest statement All authors have no conflict of interest to report. Acknowledgments This study was partly funded by grants from the Italian Association for Cancer Research (AIRC) and the “Programma Integrato Oncologia (RO4/2007)”. The authors would like to thank Emma D’Andrea and Luigi Chieco-Bianchi for their critical reading of the manuscript. They also thank Rogatus Kabyemera of the Department of Pediatrics and Child Health, Bugando Medical Centre, Mwanza, Tanzania, for African DBS samples. Contributions: C.M. and A.D.R. provided the conception and design of the study, drifting the article, revised it critically for important intellectual content, and final approval of the version to be submitted; M.R.P. performed the research, analyzed the data and drafting of manuscript; L.E. and M.Q. performed all experiments; M.Z. and M.C.S. contributed to the technique support; N.M. collected African samples and provided clinical annotations. All authors have read and approved the final version of the manuscript. References [1] van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT983936. Leukemia 2003;17:2257–317. [2] van Dongen JJ, Wolvers-Tettero IL. Analysis of immunoglobulin and T cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clin Chim Acta 1991;198:93–174. [3] van Krieken JH, Langerak AW, Macintyre EA, Kneba M, Hodges E, Sanz RG, et al. Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 concerted action BHM4-CT98-3936. Leukemia 2007;21:201–6. [4] Carbone A, Gloghini A, Dotti G. EBV-associated lymphoproliferative disorders: classification and treatment. Oncologist 2008;13:577–85. [5] Ometto L, Menin C, Masiero S, Bonaldi L, Del Mistro A, Cattelan AM, et al. Molecular profile of Epstein–Barr virus in human immunodeficiency virus type 1-related lymphadenopathies and lymphomas. Blood 1997;90:313–22. [6] Cader FZ, Kearns P, Young L, Murray P, Vockerodt M. The contribution of the Epstein–Barr virus to the pathogenesis of childhood lymphomas. Cancer Treat Rev 2010;36:348–53.
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