0 1992 Harwood Academic Publishers GmbH
Leukemia and Lymphoma, Vol. 7, pp. 289-295 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Variable Rate of Detection of Immunoglobulin Heavy Chain V-D-J Rearrangement by PCR: a Systematic Study of 41 B-Cell Non-Hodgki n’s Lymphomas and Leukemias
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JONATHAN BEN-EZRA’ From the Department of Pathology, City of Hope National Medical Center, Duarte, CA, USA. ‘Current address: Department of Pathology, Medical College of Virginia, Richmond, VA, USA. (Received 6 February 1992)
The use of clonospecific probes has recently been employed for the detection of minimal residual disease in B- and T-cell acute lymphoblastic leukemias. However, these methods are predicated upon the successful amplification of the V-D-J rearrangement in the genome of the tumor cells by the polymerase chain reaction (PCR). In order to determine whether the type of B-cell lymphoid malignancy influenced the rate of success of amplifying the region of the immunoglobulin heavy chain gene rearrangement in these lesions, we studied 41 morphologically and immunologically well characterized B-cell neoplasms. DNA was extracted from frozen tissue of the lymphomas and leukemias, and subjected to PCR amplification using a 5’ immunoglobulin heavy chain gene variable region consensus Framework 3 region (FR3) primer, and a 3’ consensus primer for the immunoglobulin heavy chain joining region. One or two distinct bands, representing the rearranged immunoglobulin heavy chain gene, were detected in six of six small non-cleaved cell lymphomas, five of five small lymphocytic lymphomas, four of six acute lymphoblastic leukemias, four of six follicular lymphomas, three of six diffuse mixed small and large cell lymphomas, one of six diffuse large cell lymphomas, and one of six immunoblastic large cell lymphomas; all control cases of lymphocyte predominant Hodgkin’s disease (5/5) and reactive follicular hyperplasia ( 5 / 5 ) were negative. We therefore conclude that the type of B-cell neoplasm influences the ability to detect immunoglobulin gene rearrangements by PCR with currently used consensus primers. KEY WORDS:
Gene rearrangement
methods
INTRODZJCTION In the course of normal lymphoid development, the progenitor lymphoid cell undergoes somatic gene rearrangement, whereby the variable (V), joining (J), Address for correspondence: Dr. Jonathan Ben-Ezra, Department of Pathology, Medical College of Virginia, Box 597, Richmond, VA, 23298-0597, USA. Address reprints to: B and T Laboratory, Department of Pathology, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA.
non-Hodgkin’s lymphoma
PCR
and diversity (D) regions of a T-cell antigen receptor and/or B-cell immunoglobulin gene undergo a unique reorganization’.’. Whereas these regions are normally kilobases apart in the genome, the rearrangement brings a functional variable region within 200 bp of a joining region3. Since a T- or B-cell lymphoid malignancy is a clonal proliferation of lymphoid cells, all of the cells in a lymphoma or lymphoid leukemia share the same immunoglobulin or T-cell receptor gene rearrangement. Detection of this rearrangement by Southern hybridization has been used throughout 289
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the past decade for the detection of clonality in lymphoid neoplasms4. However, for the purposes of detecting minimal residual disease (MRD), this method is poor, since inherent technical considerations of a Southern blot limit its sensitivity to only approximately 1% (one clonal cell in 100 cells). In the past several years, an exquisitely sensitive molecular biology technique, polymerase chain reaction (PCR), has been developed5. With this technique, one can detect a particular DNA or RNA sequence in as few as one in 100,000 cells. In hematopathology, this technique has been most frequently used to detect chromosomal translocations, such as the t(14;18) translocation present in follicular lymphomas, where one is searching for a genomic sequence not normally found in human tissues6. Recently, this technique has been applied to detecting the B - ~ e l l ~ - ' -or ' ~ T-cell' 8-z4 gene rearrangements which are present in lymphoid malignancies, and has been successfully employed in detecting MRD in these neoplasms. However, to successfully detect MRD, one has to be able to amplify the V-D-J rearrangement by PCR. Although a few studies have studied a spectrum of B-cell lymphomas with this technique7*'3 * 1', only one has specifically mentioned the types of lymphomas which were e ~ a m i n e d ' ~and , no systematic study of the amplifying the V-D-J rearrangement of various B-cell lymphomas and leukemias by PCR has been undertaken. It was the purpose of this study to undertake such an investigation, to determine if the immunoglobulin heavy chain rearrangement was more commonly detectable by PCR in some types of lymphoid tumors as compared to others.
MATERIALS All of the cases for this study were culled from the research files and frozen tumor bank of the Hematopathology Laboratory, Division of Pathology, City of Hope National Medical Center, Duarte, CA, USA. For the purposes of this study, six cases each of follicular lymphoma, diffuse mixed small and large cell lymphoma, diffuse large ("histiocytic") cell lymphoma, diffuse large cell immunoblastic lymphoma, small non-cleaved cell lymphoma, and acute
lymphoblastic leukemia (ALL), and five cases each of small (well-differentiated) lymphocytic lymphoma, lymphocyte predominant Hodgkin's disease, and reactive follicular hyperplasia were studied; the latter two categories served as negative controls. All of the cases had been studied morphologically and had been immunologically phenotyped by well-established techniquesz5, and were determined to be of B-cell lineage.
METHODS AND RESULTS DNA extraction and gene rearrangement studies Extraction of DNA from the frozen tissue hlocks and cell suspensions was performed as previously describedz6. For the gene rearrangement studies, 10 p g of DNA was digested with either Bum HI, Eco RI, or Hind 111, run on a 0.7% agarose gel, transferred to a nylon membrane, and probed for rearrangements of the immunoglobulin heavy chain gene according to previously described protocolsz7;the genomic probe for the immunoglobulin heavy chain region was a generous gift of Dr. Philip Lederz8.
PCR amplification Consensus primers for the Framework 3 (FR3) portion of the variable region and of the 3' portion of the joining region of the immunoglobulin heavy chain gene (Table l), based on previously published studies7*'5 , were obtained from a commercial source (Operon Technologies Inc., Alameda, CA). The amplification reaction was performed in 50 pl of the manufacturer's (Perkin-Elmer Cetus Corporation, Norwalk, CT) 1X buffer which contained 100 ng of template DNA, 50 picomoles of each primer, 200 pM of each dNTP, 3.5 mM MgCl,, and 1.25 U of Tuq DNA polymerase (Perkin-Elmer Cetus Corporation, Norwalk, CT). The reaction consisted of 30 cycles on a programmable thermal cycler (Perkin-Elmer Cetus Corporation) of a denaturation stem at 93°C for 2 minutes (95OC for the first cycle), an annealing step at 55°C for 30 sec, and an extension step for 30 sec at 72°C (10 minutes last cycle). The reaction products were separated on a 4% agarose gel and transferred
Table 1 Primers used in PCR reaction FR3 Variable region consensus primer Consensus joining region primer Internal Probe (joining region)
ACA CGG C[C/T][G/C] TGT A T T ACT GT ACC TGA GGA GAC GGT GAC C GTG ACC AGG GTN CCT TGG CCC CAG
Ig PCR IN NHL AND LEUKEMIAS
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to a nylon membrane (Zetaprobe, Bio-Rad Laboratories, Richmond, CA), after the gel had been stained with ethidium bromide and analyzed under ultraviolet light. The filters were then hybridized with an internal oligonucleotide probe corresponding to the joining region of the immunoglobulin heavy chain gene (Table 1). The filters were washed, and exposed overnight to x-ray film in cassettes with intensifying screens at -70°C. Tubes with placental DNA as template as well as reaction tubes without any DNA template were utilized as negative controls in each experiment to detect possible contamination. Results The results of our ability to amplify the V-D-J rearrangement of the immunoglobulin heavy chain gene by PCR are summarized in Table 2. Clear differences were present between groups, with success rates varying from 100% for small (“well-differentiated”) lymphocytic lymphoma and small non-
29 1
cleaved cell lymphoma, to 17% for large cell lymphomas. Overall, we obtained at least one distinct band representing a rearrangement in 24 of 41 cases (58%). For the positive samples, one, and sometimes two, sharp bands were present between 90 and 130 bp in length (Figures 1 and 2). In some of the negative samples and in the reactive follicular hyperplasia controls, a broad streak without discrete bands, representing a polyclonal population of cells’ 5 , was present. The reactions with placental DNA as template, as well as those tubes without DNA template, were consistently negative (Figures 1 and 2). Although we had initially selected our cases based on a B-cell immunophenotype as determined by immunohistochemical studies, the possibility remained that these tumors were in fact not B-cell
Table 2 Success of PCR to detect immunoglobulin heavy chain rearrangements Lesion
Small lymphocytic lymphoma Follicular lymphoma Diffuse small and large cell lymphoma Diffuse large cell lymphoma Diffuse large cell immunoblastic lymphoma Small non-cleaved cell lymphoma Acute lymphoblastic leukemia Lymphocyte Predominant Hodgkin’s Disease, Nodular Reactive follicular hyperplasia
Cases ampli$ed/iotal
515 416 3/6 116 1i6 616 4i6
015 015
Figure I PCR analysis of heavy chain rearrangement. The procedure was performed as detailed in the Methods section. The samples were from small lymphocytic lymphoma (lanes l-5), reactive follicular hyperplasia (lanes 6 and 7), placental DNA (lane 8) and no DNA (negative water control-lane 9). Relevant size markers are as given.
Figure 2 PCR analysis of heavy chain rearrangement. The samples were from diffuse mixed small and large cell lymphoma (lanes 1 4 ) , large cell immunoblastic lymphoma (lane 5-7). diffuse large cell lymphoma (lanes 8-1 1). nodular (follicular) lymphoma (lanes 12- 14). placental DNA (lane 15) and no DNA (negative water control-lane 16). Notice the lack of a distinct band in lanes 1,5,7,8,9,11 and 1 3
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added to or deleted from the junctional sites (N regions), further adding to the uniqueness of a particular gene rearrangement for a specific B cell. This unique and distinctive gene may then be used to manufacture the antibody or surface immunoglobulin associated with that particular B lineage cell. Since a B-cell neoplasm is a clonal proliferation of cells, all the cells in that tumor will share the unique gene rearrangement of the clonal progenitor cell, and, if the cells manufacture immunoglobulin, will also produce the same immunoglobulin. This clonal identity of the unique gene rearrangement and/or immunoglobulin can be used to treat the lymphoid neoplasm (e.g. anti-idiotype antibodies), or to monitor the presence of residual disease (e.g. clonal excess, gene rearrangements, etc.). This unique rearrangement has also been utilized to detect minimal residual disease (MRD) by PCR technology. In these studies, a universal primer for the variable region and joining region of the immunoglobulin heavy chain gene are synthesized, and a product from the V-D-J rearrangement is then amplified and sequenced’ A clonospecific probe is then manufactured, being complementary to the N and D regions of the unique rearrangement, and this oligonucleotide is used to monitor the presence of MRD. However, the success of this technique is predicated upon being initially able to amplify the immunoglobulin heavy chain gene rearrangement. Unfortunately, few studies have examined a wide variety of lesions to determine if this technique is universally applicable to B-cell leukemias and lymphomas. Trainor et al. studied 14 non-Hodgkin’s lymphomas and nine cases of chronic lymphocytic leukemia, and were able to amplify, respectively, 12 (86%) and 7 (78%) cases successfully; however, they did not mention the histology of their non-Hodgkin’s lymphoma cases. Similarly, the same group’ was able to obtain positive results from 24 of 26 (92%) paraffin embedded cases of B-cell lymphoma, but, once again, no mention was made of histology. McCarthy et ul. l 3 studied nine cases of B-cell lymphoma, and obtained positive PCR results in 0 of one diffuse large cell lymphomas, three of three small lymphocytic lymphomas, one of one diffuse small cleaved cell lymphomas, and three of four follicular lymphomas, for an overall success rate of 78% (seven of nine). Although they did not look at a wide variety of cases, Jonsson et ul. were able to successfully use the PCR technique to amplify 10 of 12 cases of ALL (83%), and Liang et al. l 2 successfully amplified 14 of 17 cases of ALL (82%) and all 15 cases of chronic lymphocytic 5916.
Figure 3 Autoradiograms of DNA digested with Bum H1 and hybridizied with the heavy chain joining region probe. The case numbers correspond to the same numbered samples in Figure 2. Although gene rearrangements were detected in all three samples by Southern blotting, only one of the specimens (lane 6 ) had detectable gene rearrangements by PCR analysis.
’
neoplasms, or that they lacked immunoglobulin heavy chain gene rearrangements. We therefore performed genomic Southern hybridization studies to demonstrate immunoglobulin heavy chain gene rearrangements in our cases. All of our B-cell neoplasms contained a rearranged band in at least one restriction enzyme digest (Figure 3), whereas none of the reactive or Hodgkin’s disease controls displayed such genomic reorganization upon probing with the immunoglobulin heavy chain joining region probe.
DISCUSSION During its development, every B cell undergoes a unique gene rearrangement, where one of many variable (V), diversity (D), and joining (J) region genes are brought into proximity to make a functional gene’. During this rearrangement, nucleotides are
’
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Ig PCR IN NHL AND LEUKEMIAS
leukemia (CLL) which they studied (100%).Although slightly different amplification conditions were employed in each of these reports, all of the groups used similar, if not identical, FR3 (5’) and joining (3’) region primers. In this study, we analyzed a wide variety of B-cell neoplasms, and were able to successfully amplify 58% of these tumors (Table 2). One question that should be asked is why weren’t we in this study, and other investigators in other report^^.'^*'^,'^*^^ , able to amplify all the B-cell neoplasms studied? One explanation might be that different tumors preferentially use different variable region gene families for rearrangements, and the “universal” primers used may not detect all of these families. As an example, ALLs and CLLs preferentially use the VH families most proximal to the IgH joining locus”. Of interest is the fact that these two lesions were among the most successfully amplified types of tumors in this report [small lymphocytic lymphoma is the tissue equivalent of CLL29] and in other reports. However, these primers were designed to have over 85% homology with >95% of the variable FR3 sequence^'^-'^, and therefore should be able to amplify almost all V-D-J rearrangements. Alternatively, the variable region may not in all B-cell tumors be brought into proximity to the joining region of the immunoglobulin heavy chain gene, either because only D-J, without the full V-D-J, rearrangement, has occurred, or because the rearrangement has been disrupted by a chromosomal translocation ”. Compared to other studies, our overall success rate of 58% appears poor. Although one may be tempted to speculate that not all of the cases we studied were of B-cell lineage, we do not believe that this is the case. Using an extensive panel of immunologic markers, all of these tumors had been previously characterized as being of B-cell derivation. Moreover, we performed genomic Southern hybridization gene rearrangement studies to confirm that a heavy chain rearrangement was indeed present. This was done to avoid results similar to that obtained by Liang et a l l 2 where neither of two cases of B-lineage ALL which lacked heavy chain gene rearrangements by genomic studies had an amplifiable product on PCR studies. The sensitivity of the PCR technique should not be an issue, since, as used in this study, the technique has a sensitivity of approximately 5%’, similar to that of the positive genomic hybridizations studies which we obtained. Inadequacy of the internal oligonucleotide probe cannot be invoked to explain the negative results, since the ethidium bromide stained gels also
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failed to reveal bands in lanes of the negative specimens. Moreover, our technique does not appear to be at fault, since our negative controls were appropriately negative, and we had better success in those types of lesions, such as ALL, small lymphocytic lymphoma, and follicular lymphoma, with which other groups also had superior result^^-'^*'^"^ . W e are therefore left with the conclusion that it was the case mix used in this study, with some types of B-cell tumors being amplified more readily than others, that was the cause of our “poor” results of being to successfully amplify only 58% of the tumors we studied. In this study, our negative controls included cases of reactive follicular hyperplasia, placental DNA as template, no template (“water”) controls, and cases of nodular lymphocyte predominant Hodgkin’s disease. This latter group was included to determine if they were indeed B-cell neoplasms. Several immunologic30-32 and p a t h ~ l o g i cstudies ~ ~ have indicated that this subtype of Hodgkin’s disease may be of B-cell derivation. Although one is not able to detect this with conventional gene rearrangement studies, since the quantity of neoplastic cells is so small, it was our hope that, if a clonal B-cell proliferation did exist, that this clone (or clones) would comprise more than 5% of the B-cell presents, since the reactive T-cells present in the background of the lesions would not add to the background product in the PCR amplified material, which is only comprised of B-cells. Our lack of successful amplification with this group of tumors does not distinguish between the possibilities that; (1) nodular lymphocyte predominant Hodgkin’s disease is not derived from a clonal proliferation of B cells, or; (2) if a clone of B cells is present, it comprises less than 5% of the total number of B cells present in the lymph node. Molecular biology techniques, including PCR, are excellent tools for determining clonality of lymphoid neoplasms by detecting their V-D-J rearrangements. These methods may also be extended to paraffin embedded material’ 7 * 2 7 to detect clonality in archival specimens. The exquisite sensitivity of the PCR method has made it possible to clinically detect and monitor MRD in ALLl4*I5.However, the data from this study indicate that this methodology may not be applicable to all B-cell neoplasms, since some types of lymphomas, such as large cell lymphomas, were not as readily amplified as were ALLs and small lymphocytic lymphomas. Until a PCR method is shown to be truly “universal”, a negative result for the V-D-J rearrangement by PCR should not be taken
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to indicate with certainty that the lesion being studied 14. Yamada, M., Wasserman, R., Lange, B., Reichard, B. A., Womer, R. B. and Rovera, G . (1990) Minimal residual disease lacks such heavy chain rearrangements. addition,, in childhood B-lineage lymphoblastic leukemia. Persistence of if One wishes to use this technique to detect and leukemic cells during the first 18 months of treatment. N . Engl. monitor MRD, one may have to be selective in the J. Med., 323,448455. B-cell lymphomas and leukemias which are studied. 15. Yamada, M., Hudson, S., Tournay, o.,Bittenbender, s..Shane. Acknowledgements The author wishes to acknowledge the excellent technical help of Mr. Nathan Cook and Mr. Wengang Chen. He also thanks Dr. Michael Kornstein for reviewing the manuscript.
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REFERENCES 1. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature, 302, 575-581. 2. Chien, Y., Gascoigne, N. R. J., Kavaler, J., Lee, N. E. and Davis, M. M. (1984)Somatic recombination in a murine T cell receptor gene. Nuture, 309,322-326. 3. Yamada, M., Wasserman, R., Reichard, B. A., Shane, S., Caton, A. J. and Rovera, G. (1991) Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J. Exp. Med., 173, 395407. 4. Knowles 11, D. M., Pelicci, P.-G. and Dalla-Favera, R. (1987) lmmunoglobulin and T cell receptor beta chain gene DNA probes in the diagnosis and classification of human lymphoid malignancy. Molecular and Cellular Probes, 1, 15-31. 5 Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed reaction. Methods Enzymol, 155, 335-350. 6. Crescenzi, M., Seto, M., Herzig, G. P., Weiss, P. D., Griffith, R. C. and Korsmeyer, S. J. (1988) Thermostable DNA polymerase chain amplification of t( 1438) chromosome breakpoints and detection of minimal residual disease. Proc. Natl. Acad. Sci. USA, 85, 48694873. 7. Trainor, K. J., Brisco, M. J., Story, C. J. and Morley, A. A. (1990) Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood, 75, 222C2222. 8. Deane, M. and Norton, J. D. (1990) Immunoglobulin heavy chain variable region family usage is independent of tumor cell phenotype in human B lineage leukemias. Eur. J. Immunol., 20, 2209-22 17. 9. Deane, M. and Norton, J. D. (1990) Detection of immunoglobulin gene rearrangement in B lymphoid malignancies by polymerase chain reaction gene amplification. Br. J. Haematol., 74, 251-256. 10. Brisco, M. J., Tan, L. W., Orsborn, A. M. and Morley, A. A. (1990) Development of a highly sensitive assay, based on the polymerase chain reaction, for rare B-lymphocyte clones in a polyclonal population. Br. J. Haematol., 75, 163-167. 11. Nizet, Y., Martiat, P., Vaerman, J. L., Philippe, M., Wildmann, C., Staelens, J. P., Cornu, G., Ferrant, A., Michaux, J. L. and Sokal, G. (1991) Follow-up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome. Br. J. Haematol., 79, 205-210. 12. Liang, R., Chan, V., Chan, T. K., Wong, T., Chiu, E. and Todd, D. (1991) Detection of immunoglobulin gene rearrangement in acute and chronic lymphoid malignancies of B-cell lineage by polymerase chain reaction gene amplification. Am. J. Hemato/., 38, 189-193. 13. McCarthy, K. P., Sloane, J. P. and Wiedemann, L. M. (1990) Rapid method for distinguishing clonal from polyclonal B cell populations in surgical biopsy specimens. J. Clin.Patho/., 43, 429432.
S. S., Lange, B., Tsujimoto, Y., Caton, A. J. and Rovera, G . (1989) Detection of minimal residual disease in hematopoietic malignancies of the B-cell lineage by using third-complementary-determining region (CDR-111)-specificprobes. Proc. Natl. Acad. Sci. USA, 86, 5123-5127. 16. Jonsson, 0. G., Kitchens, R. L., Scott, F. C. and Smith, R. G. (1990) Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes. Blood, 76, 2072-2079. 17. Wan, J. H., Trainor, K. J., Brisco, M. J. and Morley, A. A. (1990) Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction. J. Clin. Pathol., 43, 888-890. 18. Macintyre, E., d’Auriol, L., Amesland, F., Loiseau, P., Chen, Z., Boumsell, L., Galibert, F. and Sigaux, F. (1989) Analysis of junctional diversity in the preferential V61LJ61 rearrangement of fresh T-acute lymphoblastic leukemia cells by in vitro gene amplification and direct sequencing. Blood, 74, 2053-2061. 19. Macintyre, E. A., d’Auriol, L., Duparc, N., Leverger, G . , Galibert, F. and Sigaux, F. (1990) Use of oligonucleotide probes directed against T cell antigen receptor gamma delta variable-(diversity)-joining junctional sequences as a general method for detecting minimal residual disease in acute lymphoblastic leukemias. J. Clin. Invest., 86, 212552135, 20. Campana, D., Yokota, S., Coustan-Smith, E., Hansen-Hagge, T. E., Janossy, G. and Bartram, C. R. (1990) The detection of residual acute lymphoblastic leukemia cells with immunologic methods and polymerase chain reaction: A comparative study. Leukemia, 4, 609-614. 21. McCarthy, K. P., Sloane, J. P., Kabarowski, J. H. S., Matutes, E. and Wiedemann, L. M. (1991) The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders. Am. J. Pathol., 138, 821-828. 22. Broeren, C. P. M., Verjans, G. M. G. M., Van Eden, W., Kusters, J. G., Lenstra, J. A. and Logtenberg, T. (1991) Conserved nucleotide sequences at the 5’ end of the T cell receptor variable genes facilitate polymerase chain reaction amplification. Eur. J . Immunol., 21, 569-575. 23. Hansen-Hagge, T. E., Yokota, S. and Bartram, C. R. (1989) Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor 6 chain sequences. Blood, 74, 1762-1767. 24. Yokota, S., Hansen-Hagge, T. E., Ludwig, W.-D., Reiter, A., Raghavachar, A., Kleihauer, E. and Bartram, C. R. (1991) Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients. Blood, 77, 331-339. 25. Sheibani, K. and Tubbs, R. R. (1984) Enzyme immunohistochemistry: Technical aspects. Sem. Diag. Pathol., 1, 235-250. 26. Ben-Ezra, J. Winberg, C. D., Wu, A., Sheibani, K. and Rappaport, H. (1987) Concurrent presence of two clonal populations in small lymphocytic lymphoma of the lung. Hum. Pathol., 18, 399402. 27. Wu, A. M., Ben-Ezra, J., Winberg, C., Colombero, A. M. and Rappaport, H. (1990) Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffinembedded specimens. Lab. Invest., 63, 107-1 14. 28. Ravetch, J. V., Siebenlist, U., Korsmeyer, S., Waldmann, T. and Leder, P. (1981) Structure of the human immunoglobulin p locus: Characterization of embryonic and rearranged J and D genes. Cell, 27, 583-591. 29. Pangalis, G. A., Nathwani, B. N. and Rappaport, H. (1977) Malignant lymphoma, well differentiated type: Its relationship
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with chronic lymphocytic leukemia and macroglobulinemia of Waldenstrom. Cancer, 39, 999-1010. 30. Hansmann, M.-L., Stein, H., Dallenbach, F. and Fellbaum, C. (1991) Diffuse lymphocyte-predominant Hodgkin’s disease (diffuse paragranuloma): A variant of the B-cell derived nodular type. Am. J . Pathol., 138. 29-36. 31. Coles, F. B., Cartun, R. W. and Pastuszak, W. T. (1988) Hodgkin’s disease, lymphocytes-predominant type: Immunoreactivity with B-cell antibodies. Mod. Pathol., 1, 274-278.
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32. Chittal, S. M., Alard, C., Rossi, J.-F., Al Saati, T., Le Tourneau, A,, Diebold, J. and Delsol, G. (1990) Further phenotypic evidence that nodular, lymphocyte-predominant Hodgkin’s disease is a large B-cell lymphoma in evolution. Am. J. Surg. Parhol., 14, 10241035. 33. Sundeen, J. T., Cossman, J. and Jaffe, E. S . (1988) Lymphocyte predominant Hodgkin’s disease nodular subtype with coexistent “large cell lymphoma”-Histological progression or composite malignancy‘? Am. J. Surg. Pathol., 12, 599-606.
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Variable Rate of Detection of Immunoglobulin Heavy Chain V-D-J Rearrangement by PCR: a Systematic Study of 41 B-Cell Non-Hodgki n’s Lymphomas and Leukemias
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JONATHAN BEN-EZRA’ From the Department of Pathology, City of Hope National Medical Center, Duarte, CA, USA. ‘Current address: Department of Pathology, Medical College of Virginia, Richmond, VA, USA. (Received 6 February 1992)
The use of clonospecific probes has recently been employed for the detection of minimal residual disease in B- and T-cell acute lymphoblastic leukemias. However, these methods are predicated upon the successful amplification of the V-D-J rearrangement in the genome of the tumor cells by the polymerase chain reaction (PCR). In order to determine whether the type of B-cell lymphoid malignancy influenced the rate of success of amplifying the region of the immunoglobulin heavy chain gene rearrangement in these lesions, we studied 41 morphologically and immunologically well characterized B-cell neoplasms. DNA was extracted from frozen tissue of the lymphomas and leukemias, and subjected to PCR amplification using a 5’ immunoglobulin heavy chain gene variable region consensus Framework 3 region (FR3) primer, and a 3’ consensus primer for the immunoglobulin heavy chain joining region. One or two distinct bands, representing the rearranged immunoglobulin heavy chain gene, were detected in six of six small non-cleaved cell lymphomas, five of five small lymphocytic lymphomas, four of six acute lymphoblastic leukemias, four of six follicular lymphomas, three of six diffuse mixed small and large cell lymphomas, one of six diffuse large cell lymphomas, and one of six immunoblastic large cell lymphomas; all control cases of lymphocyte predominant Hodgkin’s disease (5/5) and reactive follicular hyperplasia ( 5 / 5 ) were negative. We therefore conclude that the type of B-cell neoplasm influences the ability to detect immunoglobulin gene rearrangements by PCR with currently used consensus primers. KEY WORDS:
Gene rearrangement
methods
INTRODZJCTION In the course of normal lymphoid development, the progenitor lymphoid cell undergoes somatic gene rearrangement, whereby the variable (V), joining (J), Address for correspondence: Dr. Jonathan Ben-Ezra, Department of Pathology, Medical College of Virginia, Box 597, Richmond, VA, 23298-0597, USA. Address reprints to: B and T Laboratory, Department of Pathology, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA.
non-Hodgkin’s lymphoma
PCR
and diversity (D) regions of a T-cell antigen receptor and/or B-cell immunoglobulin gene undergo a unique reorganization’.’. Whereas these regions are normally kilobases apart in the genome, the rearrangement brings a functional variable region within 200 bp of a joining region3. Since a T- or B-cell lymphoid malignancy is a clonal proliferation of lymphoid cells, all of the cells in a lymphoma or lymphoid leukemia share the same immunoglobulin or T-cell receptor gene rearrangement. Detection of this rearrangement by Southern hybridization has been used throughout 289
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the past decade for the detection of clonality in lymphoid neoplasms4. However, for the purposes of detecting minimal residual disease (MRD), this method is poor, since inherent technical considerations of a Southern blot limit its sensitivity to only approximately 1% (one clonal cell in 100 cells). In the past several years, an exquisitely sensitive molecular biology technique, polymerase chain reaction (PCR), has been developed5. With this technique, one can detect a particular DNA or RNA sequence in as few as one in 100,000 cells. In hematopathology, this technique has been most frequently used to detect chromosomal translocations, such as the t(14;18) translocation present in follicular lymphomas, where one is searching for a genomic sequence not normally found in human tissues6. Recently, this technique has been applied to detecting the B - ~ e l l ~ - ' -or ' ~ T-cell' 8-z4 gene rearrangements which are present in lymphoid malignancies, and has been successfully employed in detecting MRD in these neoplasms. However, to successfully detect MRD, one has to be able to amplify the V-D-J rearrangement by PCR. Although a few studies have studied a spectrum of B-cell lymphomas with this technique7*'3 * 1', only one has specifically mentioned the types of lymphomas which were e ~ a m i n e d ' ~and , no systematic study of the amplifying the V-D-J rearrangement of various B-cell lymphomas and leukemias by PCR has been undertaken. It was the purpose of this study to undertake such an investigation, to determine if the immunoglobulin heavy chain rearrangement was more commonly detectable by PCR in some types of lymphoid tumors as compared to others.
MATERIALS All of the cases for this study were culled from the research files and frozen tumor bank of the Hematopathology Laboratory, Division of Pathology, City of Hope National Medical Center, Duarte, CA, USA. For the purposes of this study, six cases each of follicular lymphoma, diffuse mixed small and large cell lymphoma, diffuse large ("histiocytic") cell lymphoma, diffuse large cell immunoblastic lymphoma, small non-cleaved cell lymphoma, and acute
lymphoblastic leukemia (ALL), and five cases each of small (well-differentiated) lymphocytic lymphoma, lymphocyte predominant Hodgkin's disease, and reactive follicular hyperplasia were studied; the latter two categories served as negative controls. All of the cases had been studied morphologically and had been immunologically phenotyped by well-established techniquesz5, and were determined to be of B-cell lineage.
METHODS AND RESULTS DNA extraction and gene rearrangement studies Extraction of DNA from the frozen tissue hlocks and cell suspensions was performed as previously describedz6. For the gene rearrangement studies, 10 p g of DNA was digested with either Bum HI, Eco RI, or Hind 111, run on a 0.7% agarose gel, transferred to a nylon membrane, and probed for rearrangements of the immunoglobulin heavy chain gene according to previously described protocolsz7;the genomic probe for the immunoglobulin heavy chain region was a generous gift of Dr. Philip Lederz8.
PCR amplification Consensus primers for the Framework 3 (FR3) portion of the variable region and of the 3' portion of the joining region of the immunoglobulin heavy chain gene (Table l), based on previously published studies7*'5 , were obtained from a commercial source (Operon Technologies Inc., Alameda, CA). The amplification reaction was performed in 50 pl of the manufacturer's (Perkin-Elmer Cetus Corporation, Norwalk, CT) 1X buffer which contained 100 ng of template DNA, 50 picomoles of each primer, 200 pM of each dNTP, 3.5 mM MgCl,, and 1.25 U of Tuq DNA polymerase (Perkin-Elmer Cetus Corporation, Norwalk, CT). The reaction consisted of 30 cycles on a programmable thermal cycler (Perkin-Elmer Cetus Corporation) of a denaturation stem at 93°C for 2 minutes (95OC for the first cycle), an annealing step at 55°C for 30 sec, and an extension step for 30 sec at 72°C (10 minutes last cycle). The reaction products were separated on a 4% agarose gel and transferred
Table 1 Primers used in PCR reaction FR3 Variable region consensus primer Consensus joining region primer Internal Probe (joining region)
ACA CGG C[C/T][G/C] TGT A T T ACT GT ACC TGA GGA GAC GGT GAC C GTG ACC AGG GTN CCT TGG CCC CAG
Ig PCR IN NHL AND LEUKEMIAS
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to a nylon membrane (Zetaprobe, Bio-Rad Laboratories, Richmond, CA), after the gel had been stained with ethidium bromide and analyzed under ultraviolet light. The filters were then hybridized with an internal oligonucleotide probe corresponding to the joining region of the immunoglobulin heavy chain gene (Table 1). The filters were washed, and exposed overnight to x-ray film in cassettes with intensifying screens at -70°C. Tubes with placental DNA as template as well as reaction tubes without any DNA template were utilized as negative controls in each experiment to detect possible contamination. Results The results of our ability to amplify the V-D-J rearrangement of the immunoglobulin heavy chain gene by PCR are summarized in Table 2. Clear differences were present between groups, with success rates varying from 100% for small (“well-differentiated”) lymphocytic lymphoma and small non-
29 1
cleaved cell lymphoma, to 17% for large cell lymphomas. Overall, we obtained at least one distinct band representing a rearrangement in 24 of 41 cases (58%). For the positive samples, one, and sometimes two, sharp bands were present between 90 and 130 bp in length (Figures 1 and 2). In some of the negative samples and in the reactive follicular hyperplasia controls, a broad streak without discrete bands, representing a polyclonal population of cells’ 5 , was present. The reactions with placental DNA as template, as well as those tubes without DNA template, were consistently negative (Figures 1 and 2). Although we had initially selected our cases based on a B-cell immunophenotype as determined by immunohistochemical studies, the possibility remained that these tumors were in fact not B-cell
Table 2 Success of PCR to detect immunoglobulin heavy chain rearrangements Lesion
Small lymphocytic lymphoma Follicular lymphoma Diffuse small and large cell lymphoma Diffuse large cell lymphoma Diffuse large cell immunoblastic lymphoma Small non-cleaved cell lymphoma Acute lymphoblastic leukemia Lymphocyte Predominant Hodgkin’s Disease, Nodular Reactive follicular hyperplasia
Cases ampli$ed/iotal
515 416 3/6 116 1i6 616 4i6
015 015
Figure I PCR analysis of heavy chain rearrangement. The procedure was performed as detailed in the Methods section. The samples were from small lymphocytic lymphoma (lanes l-5), reactive follicular hyperplasia (lanes 6 and 7), placental DNA (lane 8) and no DNA (negative water control-lane 9). Relevant size markers are as given.
Figure 2 PCR analysis of heavy chain rearrangement. The samples were from diffuse mixed small and large cell lymphoma (lanes 1 4 ) , large cell immunoblastic lymphoma (lane 5-7). diffuse large cell lymphoma (lanes 8-1 1). nodular (follicular) lymphoma (lanes 12- 14). placental DNA (lane 15) and no DNA (negative water control-lane 16). Notice the lack of a distinct band in lanes 1,5,7,8,9,11 and 1 3
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added to or deleted from the junctional sites (N regions), further adding to the uniqueness of a particular gene rearrangement for a specific B cell. This unique and distinctive gene may then be used to manufacture the antibody or surface immunoglobulin associated with that particular B lineage cell. Since a B-cell neoplasm is a clonal proliferation of cells, all the cells in that tumor will share the unique gene rearrangement of the clonal progenitor cell, and, if the cells manufacture immunoglobulin, will also produce the same immunoglobulin. This clonal identity of the unique gene rearrangement and/or immunoglobulin can be used to treat the lymphoid neoplasm (e.g. anti-idiotype antibodies), or to monitor the presence of residual disease (e.g. clonal excess, gene rearrangements, etc.). This unique rearrangement has also been utilized to detect minimal residual disease (MRD) by PCR technology. In these studies, a universal primer for the variable region and joining region of the immunoglobulin heavy chain gene are synthesized, and a product from the V-D-J rearrangement is then amplified and sequenced’ A clonospecific probe is then manufactured, being complementary to the N and D regions of the unique rearrangement, and this oligonucleotide is used to monitor the presence of MRD. However, the success of this technique is predicated upon being initially able to amplify the immunoglobulin heavy chain gene rearrangement. Unfortunately, few studies have examined a wide variety of lesions to determine if this technique is universally applicable to B-cell leukemias and lymphomas. Trainor et al. studied 14 non-Hodgkin’s lymphomas and nine cases of chronic lymphocytic leukemia, and were able to amplify, respectively, 12 (86%) and 7 (78%) cases successfully; however, they did not mention the histology of their non-Hodgkin’s lymphoma cases. Similarly, the same group’ was able to obtain positive results from 24 of 26 (92%) paraffin embedded cases of B-cell lymphoma, but, once again, no mention was made of histology. McCarthy et ul. l 3 studied nine cases of B-cell lymphoma, and obtained positive PCR results in 0 of one diffuse large cell lymphomas, three of three small lymphocytic lymphomas, one of one diffuse small cleaved cell lymphomas, and three of four follicular lymphomas, for an overall success rate of 78% (seven of nine). Although they did not look at a wide variety of cases, Jonsson et ul. were able to successfully use the PCR technique to amplify 10 of 12 cases of ALL (83%), and Liang et al. l 2 successfully amplified 14 of 17 cases of ALL (82%) and all 15 cases of chronic lymphocytic 5916.
Figure 3 Autoradiograms of DNA digested with Bum H1 and hybridizied with the heavy chain joining region probe. The case numbers correspond to the same numbered samples in Figure 2. Although gene rearrangements were detected in all three samples by Southern blotting, only one of the specimens (lane 6 ) had detectable gene rearrangements by PCR analysis.
’
neoplasms, or that they lacked immunoglobulin heavy chain gene rearrangements. We therefore performed genomic Southern hybridization studies to demonstrate immunoglobulin heavy chain gene rearrangements in our cases. All of our B-cell neoplasms contained a rearranged band in at least one restriction enzyme digest (Figure 3), whereas none of the reactive or Hodgkin’s disease controls displayed such genomic reorganization upon probing with the immunoglobulin heavy chain joining region probe.
DISCUSSION During its development, every B cell undergoes a unique gene rearrangement, where one of many variable (V), diversity (D), and joining (J) region genes are brought into proximity to make a functional gene’. During this rearrangement, nucleotides are
’
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Ig PCR IN NHL AND LEUKEMIAS
leukemia (CLL) which they studied (100%).Although slightly different amplification conditions were employed in each of these reports, all of the groups used similar, if not identical, FR3 (5’) and joining (3’) region primers. In this study, we analyzed a wide variety of B-cell neoplasms, and were able to successfully amplify 58% of these tumors (Table 2). One question that should be asked is why weren’t we in this study, and other investigators in other report^^.'^*'^,'^*^^ , able to amplify all the B-cell neoplasms studied? One explanation might be that different tumors preferentially use different variable region gene families for rearrangements, and the “universal” primers used may not detect all of these families. As an example, ALLs and CLLs preferentially use the VH families most proximal to the IgH joining locus”. Of interest is the fact that these two lesions were among the most successfully amplified types of tumors in this report [small lymphocytic lymphoma is the tissue equivalent of CLL29] and in other reports. However, these primers were designed to have over 85% homology with >95% of the variable FR3 sequence^'^-'^, and therefore should be able to amplify almost all V-D-J rearrangements. Alternatively, the variable region may not in all B-cell tumors be brought into proximity to the joining region of the immunoglobulin heavy chain gene, either because only D-J, without the full V-D-J, rearrangement, has occurred, or because the rearrangement has been disrupted by a chromosomal translocation ”. Compared to other studies, our overall success rate of 58% appears poor. Although one may be tempted to speculate that not all of the cases we studied were of B-cell lineage, we do not believe that this is the case. Using an extensive panel of immunologic markers, all of these tumors had been previously characterized as being of B-cell derivation. Moreover, we performed genomic Southern hybridization gene rearrangement studies to confirm that a heavy chain rearrangement was indeed present. This was done to avoid results similar to that obtained by Liang et a l l 2 where neither of two cases of B-lineage ALL which lacked heavy chain gene rearrangements by genomic studies had an amplifiable product on PCR studies. The sensitivity of the PCR technique should not be an issue, since, as used in this study, the technique has a sensitivity of approximately 5%’, similar to that of the positive genomic hybridizations studies which we obtained. Inadequacy of the internal oligonucleotide probe cannot be invoked to explain the negative results, since the ethidium bromide stained gels also
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failed to reveal bands in lanes of the negative specimens. Moreover, our technique does not appear to be at fault, since our negative controls were appropriately negative, and we had better success in those types of lesions, such as ALL, small lymphocytic lymphoma, and follicular lymphoma, with which other groups also had superior result^^-'^*'^"^ . W e are therefore left with the conclusion that it was the case mix used in this study, with some types of B-cell tumors being amplified more readily than others, that was the cause of our “poor” results of being to successfully amplify only 58% of the tumors we studied. In this study, our negative controls included cases of reactive follicular hyperplasia, placental DNA as template, no template (“water”) controls, and cases of nodular lymphocyte predominant Hodgkin’s disease. This latter group was included to determine if they were indeed B-cell neoplasms. Several immunologic30-32 and p a t h ~ l o g i cstudies ~ ~ have indicated that this subtype of Hodgkin’s disease may be of B-cell derivation. Although one is not able to detect this with conventional gene rearrangement studies, since the quantity of neoplastic cells is so small, it was our hope that, if a clonal B-cell proliferation did exist, that this clone (or clones) would comprise more than 5% of the B-cell presents, since the reactive T-cells present in the background of the lesions would not add to the background product in the PCR amplified material, which is only comprised of B-cells. Our lack of successful amplification with this group of tumors does not distinguish between the possibilities that; (1) nodular lymphocyte predominant Hodgkin’s disease is not derived from a clonal proliferation of B cells, or; (2) if a clone of B cells is present, it comprises less than 5% of the total number of B cells present in the lymph node. Molecular biology techniques, including PCR, are excellent tools for determining clonality of lymphoid neoplasms by detecting their V-D-J rearrangements. These methods may also be extended to paraffin embedded material’ 7 * 2 7 to detect clonality in archival specimens. The exquisite sensitivity of the PCR method has made it possible to clinically detect and monitor MRD in ALLl4*I5.However, the data from this study indicate that this methodology may not be applicable to all B-cell neoplasms, since some types of lymphomas, such as large cell lymphomas, were not as readily amplified as were ALLs and small lymphocytic lymphomas. Until a PCR method is shown to be truly “universal”, a negative result for the V-D-J rearrangement by PCR should not be taken
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to indicate with certainty that the lesion being studied 14. Yamada, M., Wasserman, R., Lange, B., Reichard, B. A., Womer, R. B. and Rovera, G . (1990) Minimal residual disease lacks such heavy chain rearrangements. addition,, in childhood B-lineage lymphoblastic leukemia. Persistence of if One wishes to use this technique to detect and leukemic cells during the first 18 months of treatment. N . Engl. monitor MRD, one may have to be selective in the J. Med., 323,448455. B-cell lymphomas and leukemias which are studied. 15. Yamada, M., Hudson, S., Tournay, o.,Bittenbender, s..Shane. Acknowledgements The author wishes to acknowledge the excellent technical help of Mr. Nathan Cook and Mr. Wengang Chen. He also thanks Dr. Michael Kornstein for reviewing the manuscript.
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