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Cytogenetic and molecular genetic studies of a patient with atypical lymphoid hyperplasia
Cytogenetic and Molecular Genetic Studies of a Patient with Atypical Lymphoid Hyperplasia Georgia Chenevix-Trench, Janet M. Cowan, Frederick G. Behm, Rakesh Goorha, Judith A. Brown, Eric H. Westin, and Uta Francke
ABSTRACT: We have karyotyped cells from a lymph node of a patient with atypical lymphoid hyperplasia. Among other clonal chromosomal abnormalities, a t(2;19) translocation was observed with breakpoints at 2p11.2 and 19q13. The genes for transforming growth factor ct and ft have been mapped to 2p11-p13 and 19q13, respectively, but Southern blot analysis did not reveal any alteration in the structure of these genes. Similarly, the kappa immunoglobulin gene, which maps to 2p11-p12 was not rearranged. In addition, Southern blot analysis using immunoglobulin and T-cell receptor genes as probes, did not demonstrate any clanality of either B or T cells. We propose that this patient represents an early, polyclonal stage of atypical hyperplasia. The chromosome changes observed may have been one of the etiologic factors causing this disorder. INTRODUCTION We report cytogenetic and molecular genetic analyses of a patient with diffuse l y m p h a d e n o p a t h y who was diagnosed as having atypical l y m p h o i d hyperplasia. Although clonal karyotypic abnormalities were suggestive of a neoplastic process, there was no evidence of a clonal rearrangement of i m m u n o g l o b u l i n genes as w o u l d be expected i n a B-cell neoplasm [1, 2], nor of T-cell receptor (TCR) genes, which w o u l d be expected in a T cell tumor [3, 4].
CASE REPORT
Patient W. M., an 80-year-old white male, was hospitalized for weakness and fatigue. There was no fever, chilis, night sweats, or weight loss. He did not have a family history of cancer. Three months before admission the patient was diagnosed as having Coombs-positive hemolytic anemia at another hospital. Physical exami-
From the Departmentsof Human Genetics (G. C-T.,J. A. B.), Pathology (F. G. B.). and Medicine (E. H. W.), Medical Collegeof Virginia,Richmond,VA, The Departmentof Human Genetics(J. M. C., U. F.), Yale University School of Medicine, New Haven, CT, and St. Judes Children's Research Hospital (F. G. B., R. G.), Memphis, TN. Address requests for reprints to Dr, G. Chenevix-Trench, Queensland Institute of Medical Research, Bramston Terrace, Herston QLD 4006, Australia. Received September 2, 1986; accepted October 22, 1986. 251 © 1987 Elsevier Science PublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet27:251-259 (1987) 0165-4608/87/$03,50
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nation revealed generalized lymphadenopathy with the largest node being 2 cm in greatest dimension. Splenomegaly was present but the liver was not enlarged. Chest x-ray was suggestive of minimal enlargement of mediastinal nodes. Abnormal laboratory results included a hemoglobin of 6.8 g/dl, positive direct Coombs test, platelet count of 108,000/ram 3, LDH of 364/L, and total bilirubin of 2.1/L. The white blood cell count was 4500/ram 3 with a differential of 80% ueutrophils, 14% lymphocytes, and 6% monocytes. Bone marrow examinations showed erythroid and myeloid hyperplasia without dysplastic features, active megakaryopoiesis, and an absence of lymphoproliferative process. In addition to the lymphoid disorder, the patient had squamous cell carcinoma of the cheek. A cervical lymph node biopsy was performed. The histopathology was suggestive, but not diagnostic, of a diffuse large cell lymphoma. A diagnosis of atypical lymphoid hyperplasia was rendered. Biopsies of axillary and inguinal lymph nodes were subsequently obtained and in these the histology was similar to the original cervical lymph node. Immunologic marker studies of cell suspensions and sections of frozen tissue of all three lymph node biopsies were performed. The results of the marker studies showed 42% kappa, 40% lambda, 60% mu, 25% Leu-1 (CD5), 20% Leu-4 (CD3), 8% Leu-2 (CD8), 15% Leu-3 (CD4), and 85% HLA-DR (Ia) marked cells. The same marker studies of frozen tissue sections showed an even distribution of kappa- and lambda-bearing large lymphocytes. Small lymphocytes were primarily clustered around small vessels and were predominantly T cells. The phenotype was consistent with a reactive process or diffuse B-cell lymphoma but did not exclude a malignant lymphoproliferation of T cells. The patient was initiated on corticosteroids and returned to the referring physician. He was lost to follow-up and died at home 1 month later of cardiac arrest. No autopsy was performed.
MATERIALS AND METHODS Samples from two biopsies were received from the patient.
Cytogenetic Analyses The first sample, taken from the cervical lymph node, was established as direct, 24hour and 72-hour cultures, with 0.16 p.M tetradecanoyl-O-phorbol-13-acetate added to the 72-hour culture. The second sample, from the axillary lymph node, was cultured for 48 hours only, with no stimulants.
Molecular Genetic Analyses For Southern blot analysis, 10 ~g of high molecular-weight DNA, extracted from the axillary lymph node biopsy, was digested to completion with the appropriate restriction enzymes. EcoRI was used for the detection of rearrangements of JN, CK, Ch immunoglobulin gene regions and for structural changes in TCR [3 and ~ genes and transforming growth factor (TGF) cx and 13 genes. BamHI was used for the detection of rearrangements of Cp~ and CK immunoglobulin genes, and of the TGF genes. The digested DNA was electrophoresed through a 0.8% agarose gel and transferred to nitrocellulose [5]. The filters were hybridized with the appropriate radiolabeled probes and washed under high stringency conditions before autoradiography. The following probes were used: Cp., JR, CK [6], and C)~ [7]; TCR [3 [8] and ~ [9]; pTGF-C17N3 [10] and pBC1 [11]. The TCR-[3 probe contains 464 nucleotides from the constant region of C[31 and 15 bases from the joining region. It hybridizes well
253
Atypical Lymphoid Hyperplasia
to both the C~1 and C~2 genes. The TCR-y gene probe contains 266 nucleotides from the constant region and 24 nucleotides from its 5' flanking region. It also hybridizes well to both C~/genes. The plasmid pTGF-C17N3 contains the coding sequence for TGF-(x and pBC1 contains a TGF-~ cDNA clone of approximately 1050 bp. RESULTS
Cytogenetic Findings Cytogenetic analysis was performed only on the second biopsy because there were no mitoses in the cultures established from the first biopsy. The modal number of chromosomes was 46. A total of 34 cells was karyotyped. The chromosomal findings observed are summarized in Table 1, and illustrated in Figures 1 and 2. Because of the presence of the t(2;19) with breakpoints at the bands to which the kappa immunoglobulin gene [12] and the transforming growth factors c( and 13 have been mapped [13, 14], we wished to determine if these genes had been rearranged. In addition, we sought to examine whether the clonal chromosome changes represented a T- or B-cell neoplasm, as would be indicated by clonal rearrangements of immunoglobulin or T-cell receptor genes.
Molecular Findings In order to determine if the patient had a clonal B-cell tumor, Southern blot analysis was performed using immunoglobulin gene probes for C~, JH, CK and C)~. Fragment sizes were as expected for a germ-line arrangement [2, 6, 7]. The results of the analysis of the heavy chain and kappa light chain genes are shown in Figure 3. Hybridization with C)~ revealed germline 19.0, 13.5, 7.3, and 5.1 kb EcoRI fragments (data not shown). No clonal rearrangements were detected. A mixing experiment showed that our Southern blot technique can detect a clonal rearrangement in 15% or fewer of cells (G. Chenevix-Trench, unpublished data). Although cell surface marker analysis suggested a B cell process, T-cell receptor gene rearrangements were examined to confirm that this was not a T-cell tumor. Southern blot hybridization experiments showed that TCR 13 and TCR y are both in germ-line configuration (Figure 4). No rearrangement of either of these genes was found, therefore, the lymphadenopathy was not the result of a T-cell tumor. To determine if the t(2;19) involved the TGF genes, the structure of these genes was also examined. There were no differences between the control DNA and the DNA from the patient's tumor with either TGF-c~ or TGF-[3 (Fig. 5). In addition to the results that are illustrated, hybridization with the TGF-[3 probe to a BamHI digest revealed two fragments of 9.4 kb and 7.5 kb. All these results are consistent with previous reports of TGF-(~ and TGF-~ fragment sizes [11].
Table 1 Karyotypic findings in patient W. M. Karyotype
Number of cells
46,XY,t(2;19)(p11.2;q13) 46,XY 46,XY,dir dup(2)(q33--~q37} 47,XY, + 9,t(2;19}(p11.2;q13)
15 10 5 4
254
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Karyotype of patient W. M. showing t(2;19)(p11.2;q13).
dlrdup 121 Figure 2 Partial karyotype of patient W. M. showing duplication of chromosome 2.
255
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Figure 3 Southern blot showing absence of immunoglobulin gene rearrangements. Germline rearrangement JH, Cp., and CK gene regions in DNA from a control patient with reactive hyperplasia (C) and patient W. M. DNA was digested with EcoRI or BamHI and hybridized to JH, C~, and CK probes. Fragment sizes are shown in kilobases.
DISCUSSION
The karyotypic changes found in this patient were interesting for several reasons. First, the presence of c]onal abnormalities in 25 of 34 cells is consistent with a neoplastic process, although the diagnosis of lymphoma could not be established by histopathology. The patient died without development or regression of the disease. No germ-line karyotype was available, but the abnormalities observed are highly unlikely to be constitutional. Second, although a t(2;19) has not been previously noted in non-Hodgkin lymphoma, the breakpoints involved in this translocation are the site of several genes that are of interest in the study of oncogenesis. The bands 2pll-p12 include the sites of the kappa light chain [12] and the TGF-c~ locus [13]. Apart from frequent involvement in Burkitt's lymphoma, breaks at 2p11-p12 have been observed in at least nine cases of non-Burkitt, non-Hodgkin lymphoma, including diffuse, large cell ]ymphoma [15]. In addition, 19q13 is the site of the TGF-[3 gene [14], and breaks at this site have been reported in at least 17 cases of non-Hodgkin lymphoma, including nine with diffuse large cell lymphoma [15]. Third, although this particular rearrangement has not been reported, the breakpoints involved in the interstitial duplication of chromosome 2, 2q33 and 2q37, have been reported previously in two and five cases of non-Hodgkin lymphoma, respectively, especially diffuse large cell lymphoma [15]. These cytogenetic results, therefore, were suggestive of a neoplastic process. In order to determine if the patient had a B-cell tumor with detectable clonal immunoglobulin rearrangements, Southern blot analysis was performed with DNA from the second biopsy using probes for Cp., JH, CK, and C)~. This did not reveal any clonal immunoglobulin rearrangements. Similarly we found no evidence of a clonal rearrangement of the T-cell receptor genes using probes for TCR-~ and -~]. The sensitivity of our Southern blotting technique was such that a clonal rearrangement in at least 15% of cells could be detected. Approximately 74% of the cells we karyo-
256
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Figure 4 Southern blot analysis showing lack of T-cell receptor [3 and ~ chain gene rearrangement. DNA was digested with EcoRI and hybridized to T-cell receptor B and "y chain gene probes. Arrows indicate the position of germ-line bands in lane 1 (10.5 and 4.5 kb for TCR-[3 and 4,0 and 2.1 kb for TCR-~/); lane 2 shows rearrangements of [3 ~nd ~ genes in a Tcell acute lymphocytic leukemia patient as a positive control; lane 3 shows lack of rearrangements in [3 or ~ chain gene in DNA from patient W. M.
typed had chromosome abnormalities, thus, if these cells were clonal T or B cells, gene rearrangements should have been detected. The negative results obtained were surprising in light of the cytogenetic data. Such extensive cytogenetic aberrations would not be expected in a n o n m a l i g n a n t disorder, although few studies addressing this possibility have been published. Did the cytogenetic abnormalities occur in lymphoid stem cells prior to immunoglobulin or T-cell receptor gene rearrangements? The lack of such rearrangements would suggest an oncogenic event at an early stage of T- or B-cell development. This explanation is unlikely, however, because 82% of the lymphocytes expressed surface i m m u n o g l o b u l i n s and the remaining 18% of cells marked as T-lymphocytes. It is unlikely that the cytogenetic abnormalities observed were in metastasizing
257
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G. Chenevix-Trench et al.
cells of the squamous cell carcinoma because metastasis of squamous carcinoma can be distinguished pathologically from atypical h y p e r p l a s i a and there was no evidence of such metastasis. Southern blot analysis d i d not reveal any rearrangement in the structure of the TGF genes. This could be because the w h o l e region has translocated without rearrangement of the BamHI or EcoRI sites. A similar event has been observed in acute myelocytic leukemia [16]. It is also worth noting that scanning of 30 kb of the cm y c gene in cases of Burkitt's l y m p h o m a k n o w n to have t(8;14) only revealed rearrangements of the c-myc locus in two of 11 cases [17]. Alternatively, while the translocation breakpoints and the TGF genes have been m a p p e d to a p p r o x i m a t e l y the same bands, they m a y be thousands of kilobases apart and, thus, uninvolved. In a d d i t i o n to our interest in the possibility that clonal rearrangements of the i m m u n o g l o b u l i n genes might be useful in the definitive diagnosis of a B-cell tumor, we were interested in the possibility that the k a p p a gene had been rearranged as a consequence of the t(2;19). However, Southern blot analysis d i d not provide any evidence for such a somatic rearrangement. The CK probe we u s e d hybridizes to only 13 kb of the k a p p a locus, thus, a translocation occurring outside this region w o u l d not be detected. Unfortunately there is no material available for in situ hybridization of any of these genes to further delineate the breakpoints of this interesting case. We propose that the cytogenetic abnormalities in this patient may have stimulated p o l y c l o n a l B-cell development. It was at this stage that the l y m p h nodes were b i o p s i e d and found to be expressing both k a p p a and l a m b d a i m m u n o g l o b u l i n chains. A clonal B-cell tumor might have arisen h a d the cells been subjected to a second "hit." A n analogy can be d r a w n w i t h African Burkitt's l y m p h o m a , in w h i c h it has been p r o p o s e d that p o l y c l o n a l B-cell activation results from chronic hypere n d e m i c malaria leading to e n h a n c e d p r e d i s p o s i t i o n to infection by Epstein-Barr virus [18]. The second " h i t " in Burkitt's l y m p h o m a is thought to be activation of the c-myc oncogene by c h r o m o s o m a l translocation. This w o u l d be in contrast to our proposal for the d e v e l o p m e n t of h y p e r p l a s i a in our patient, in w h i c h the polyclonal B-cell activation m a y have followed c h r o m o s o m a l mutations. However, it should be noted that the c h r o m o s o m e abnormalities observed in patient W. M., who was diagnosed as having atypical hyperplasia, differed from those reported in clones derived from a patient with clearly defined diffuse p o l y c l o n a l B-cell lymp h o m a [19]. Supported by NCI contract NCI-CP-21037 (J.M.C,U.F.). The TGF-ct and -6 probes were kindly provided by Dr. R. Derynck, Genentech Inc., South San Francisco, CA and the immunoglobulin probes by Dr. S. Korsmeyer. This article is #295 from the Department of Human Genetics, Richmond, VA.
REFERENCES
1. Korsmeyer SJ, Arnold A, Bakhshi A, Ravetch JV, Siebenlist U, Heiter P, Sharrow SO, LeBien TW, Kersey JH, Poplack DC, Leder P, Waldmann TA {1983): Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins. J Clin Invest 71:301-313. 2. Cleary ML, Chao J, Warnke R, Sklar J (1984}: lmmunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma. Proc Natl Acad Sci 81:593-597. 3. Minden MD, Toyanaa B, Ha K, Yanagi Y, Chin B, Gelfand E, Mak T {1985}: Somatic rearrangement of T-cell antigen receptor gene in human T-cell malignancies. Proc Natl Acad Sci 82:1224-1227.
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4. Flug F, Pelicci P-G, Bonetti F, Knowles DM, Dalla-Favera R (1985): T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms. Proc Natl Acad Sci 81:3460-3464. 5. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Molec Biol 98:503-508. 6. Bakhshi A, Minowada J, Arnold A, Cossman J, Jensen JP, Whang-Peng J, Waldmann TA, Korsmeyer SJ (1983): Lymphoid blast crises of chronic myelogenous leukemia represent stages in the development of B-cell precursors. N Engl J Med 309:826-831. 7. Arnold A, Cossman J, Bahkshi A, Jaffe ES, Waldmann TA, Korsmeyer SJ (1983): Immunoglobulin-gene rearrangements as unique clonal markers in human neoplasms. N Engl J Med 309:1593-1599. 8. Collins MKL, Tanigawa G, Kissonerghis A-M, Ritter M, Price KM, Tonegawa S, Owen MJ (1985): Regulation of T-cell receptor gene expression in human T-cell development. Proc Natl Acad Sci 82:45034507. 9. Murre C, Waldmann RA, Morton CC, Bongiovanni K, Waldmann TA, Shows TB, Seidman J (1985): Human ~ chains are rearranged in leukaemic T-cells and map to the short arm of chromosome 7. Nature 316:549-552. 10. Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV (1984): Human transforming growth factor-ct precursor structure and expression in E. coli. Cell 38:287-297. 11. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV (1985): Human transforming growth factor-13 complementary DNA sequence and expression in normal and transformed cells. Nature 316:701-705. 12. Malcolm S, Barton P, Murphy C, Ferguson-Smith MA, Bentley DL, Rabbitts TH (1982): Localization of the human immunoglobulin light chain variable region genes of chromosome 2 by in situ hybridization. Proc Natl Acad Sci 79:4957-4961. 13. Brissenden JE, Derynck R, Francke U (1985): Mapping of transforming growth factor ct gene on human chromosome 2 close to the breakpoint of the Burkitt's lymphoma t(2;8) variant translocation. Cancer Res 45:5593-5597. 14. Fujii D, Brissenden JE, Derynck R, Francke U (1986): Transforming growth factor 13 gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Molec Genet 12:281-288. 15. Mitelman F (1985): A Catalog of Chromosome Aberrations in Cancer. Alan R. Liss, NY. 16. Diaz MO, LeBeau MM, Pitha P, Rowley JD (1986): Interferon and c-ets-1 genes in the translocation (9;11)(p22;q23) in human acute monocytic leukemia. Science 231:265-267, 17. Dalla-Favera R, Martinotti S, Gallo RC, Erikson J, Croce CM (1983): Translocation and rearrangements of the c-myc oncogene locus in human undifferentiated B-cell lymphomas. Science 219:963-967. 18. Burkitt, DP (1969): The etiology of Burkitt's lymphoma. J Natl Cancer Inst 42:19-28. 19. Robinson JE, Brown N, Anidman W, Halliday K, Francke U, Robert MF, Andersson-Anvret M, Horstmann D, Miller G (1980): Diffuse polyclonal B-cell lymphoma during primary infection with Epstein-Barr virus. N Engl J Med 302:1293-1297.
ABSTRACT: We have karyotyped cells from a lymph node of a patient with atypical lymphoid hyperplasia. Among other clonal chromosomal abnormalities, a t(2;19) translocation was observed with breakpoints at 2p11.2 and 19q13. The genes for transforming growth factor ct and ft have been mapped to 2p11-p13 and 19q13, respectively, but Southern blot analysis did not reveal any alteration in the structure of these genes. Similarly, the kappa immunoglobulin gene, which maps to 2p11-p12 was not rearranged. In addition, Southern blot analysis using immunoglobulin and T-cell receptor genes as probes, did not demonstrate any clanality of either B or T cells. We propose that this patient represents an early, polyclonal stage of atypical hyperplasia. The chromosome changes observed may have been one of the etiologic factors causing this disorder. INTRODUCTION We report cytogenetic and molecular genetic analyses of a patient with diffuse l y m p h a d e n o p a t h y who was diagnosed as having atypical l y m p h o i d hyperplasia. Although clonal karyotypic abnormalities were suggestive of a neoplastic process, there was no evidence of a clonal rearrangement of i m m u n o g l o b u l i n genes as w o u l d be expected i n a B-cell neoplasm [1, 2], nor of T-cell receptor (TCR) genes, which w o u l d be expected in a T cell tumor [3, 4].
CASE REPORT
Patient W. M., an 80-year-old white male, was hospitalized for weakness and fatigue. There was no fever, chilis, night sweats, or weight loss. He did not have a family history of cancer. Three months before admission the patient was diagnosed as having Coombs-positive hemolytic anemia at another hospital. Physical exami-
From the Departmentsof Human Genetics (G. C-T.,J. A. B.), Pathology (F. G. B.). and Medicine (E. H. W.), Medical Collegeof Virginia,Richmond,VA, The Departmentof Human Genetics(J. M. C., U. F.), Yale University School of Medicine, New Haven, CT, and St. Judes Children's Research Hospital (F. G. B., R. G.), Memphis, TN. Address requests for reprints to Dr, G. Chenevix-Trench, Queensland Institute of Medical Research, Bramston Terrace, Herston QLD 4006, Australia. Received September 2, 1986; accepted October 22, 1986. 251 © 1987 Elsevier Science PublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet27:251-259 (1987) 0165-4608/87/$03,50
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nation revealed generalized lymphadenopathy with the largest node being 2 cm in greatest dimension. Splenomegaly was present but the liver was not enlarged. Chest x-ray was suggestive of minimal enlargement of mediastinal nodes. Abnormal laboratory results included a hemoglobin of 6.8 g/dl, positive direct Coombs test, platelet count of 108,000/ram 3, LDH of 364/L, and total bilirubin of 2.1/L. The white blood cell count was 4500/ram 3 with a differential of 80% ueutrophils, 14% lymphocytes, and 6% monocytes. Bone marrow examinations showed erythroid and myeloid hyperplasia without dysplastic features, active megakaryopoiesis, and an absence of lymphoproliferative process. In addition to the lymphoid disorder, the patient had squamous cell carcinoma of the cheek. A cervical lymph node biopsy was performed. The histopathology was suggestive, but not diagnostic, of a diffuse large cell lymphoma. A diagnosis of atypical lymphoid hyperplasia was rendered. Biopsies of axillary and inguinal lymph nodes were subsequently obtained and in these the histology was similar to the original cervical lymph node. Immunologic marker studies of cell suspensions and sections of frozen tissue of all three lymph node biopsies were performed. The results of the marker studies showed 42% kappa, 40% lambda, 60% mu, 25% Leu-1 (CD5), 20% Leu-4 (CD3), 8% Leu-2 (CD8), 15% Leu-3 (CD4), and 85% HLA-DR (Ia) marked cells. The same marker studies of frozen tissue sections showed an even distribution of kappa- and lambda-bearing large lymphocytes. Small lymphocytes were primarily clustered around small vessels and were predominantly T cells. The phenotype was consistent with a reactive process or diffuse B-cell lymphoma but did not exclude a malignant lymphoproliferation of T cells. The patient was initiated on corticosteroids and returned to the referring physician. He was lost to follow-up and died at home 1 month later of cardiac arrest. No autopsy was performed.
MATERIALS AND METHODS Samples from two biopsies were received from the patient.
Cytogenetic Analyses The first sample, taken from the cervical lymph node, was established as direct, 24hour and 72-hour cultures, with 0.16 p.M tetradecanoyl-O-phorbol-13-acetate added to the 72-hour culture. The second sample, from the axillary lymph node, was cultured for 48 hours only, with no stimulants.
Molecular Genetic Analyses For Southern blot analysis, 10 ~g of high molecular-weight DNA, extracted from the axillary lymph node biopsy, was digested to completion with the appropriate restriction enzymes. EcoRI was used for the detection of rearrangements of JN, CK, Ch immunoglobulin gene regions and for structural changes in TCR [3 and ~ genes and transforming growth factor (TGF) cx and 13 genes. BamHI was used for the detection of rearrangements of Cp~ and CK immunoglobulin genes, and of the TGF genes. The digested DNA was electrophoresed through a 0.8% agarose gel and transferred to nitrocellulose [5]. The filters were hybridized with the appropriate radiolabeled probes and washed under high stringency conditions before autoradiography. The following probes were used: Cp., JR, CK [6], and C)~ [7]; TCR [3 [8] and ~ [9]; pTGF-C17N3 [10] and pBC1 [11]. The TCR-[3 probe contains 464 nucleotides from the constant region of C[31 and 15 bases from the joining region. It hybridizes well
253
Atypical Lymphoid Hyperplasia
to both the C~1 and C~2 genes. The TCR-y gene probe contains 266 nucleotides from the constant region and 24 nucleotides from its 5' flanking region. It also hybridizes well to both C~/genes. The plasmid pTGF-C17N3 contains the coding sequence for TGF-(x and pBC1 contains a TGF-~ cDNA clone of approximately 1050 bp. RESULTS
Cytogenetic Findings Cytogenetic analysis was performed only on the second biopsy because there were no mitoses in the cultures established from the first biopsy. The modal number of chromosomes was 46. A total of 34 cells was karyotyped. The chromosomal findings observed are summarized in Table 1, and illustrated in Figures 1 and 2. Because of the presence of the t(2;19) with breakpoints at the bands to which the kappa immunoglobulin gene [12] and the transforming growth factors c( and 13 have been mapped [13, 14], we wished to determine if these genes had been rearranged. In addition, we sought to examine whether the clonal chromosome changes represented a T- or B-cell neoplasm, as would be indicated by clonal rearrangements of immunoglobulin or T-cell receptor genes.
Molecular Findings In order to determine if the patient had a clonal B-cell tumor, Southern blot analysis was performed using immunoglobulin gene probes for C~, JH, CK and C)~. Fragment sizes were as expected for a germ-line arrangement [2, 6, 7]. The results of the analysis of the heavy chain and kappa light chain genes are shown in Figure 3. Hybridization with C)~ revealed germline 19.0, 13.5, 7.3, and 5.1 kb EcoRI fragments (data not shown). No clonal rearrangements were detected. A mixing experiment showed that our Southern blot technique can detect a clonal rearrangement in 15% or fewer of cells (G. Chenevix-Trench, unpublished data). Although cell surface marker analysis suggested a B cell process, T-cell receptor gene rearrangements were examined to confirm that this was not a T-cell tumor. Southern blot hybridization experiments showed that TCR 13 and TCR y are both in germ-line configuration (Figure 4). No rearrangement of either of these genes was found, therefore, the lymphadenopathy was not the result of a T-cell tumor. To determine if the t(2;19) involved the TGF genes, the structure of these genes was also examined. There were no differences between the control DNA and the DNA from the patient's tumor with either TGF-c~ or TGF-[3 (Fig. 5). In addition to the results that are illustrated, hybridization with the TGF-[3 probe to a BamHI digest revealed two fragments of 9.4 kb and 7.5 kb. All these results are consistent with previous reports of TGF-(~ and TGF-~ fragment sizes [11].
Table 1 Karyotypic findings in patient W. M. Karyotype
Number of cells
46,XY,t(2;19)(p11.2;q13) 46,XY 46,XY,dir dup(2)(q33--~q37} 47,XY, + 9,t(2;19}(p11.2;q13)
15 10 5 4
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Karyotype of patient W. M. showing t(2;19)(p11.2;q13).
dlrdup 121 Figure 2 Partial karyotype of patient W. M. showing duplication of chromosome 2.
255
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Figure 3 Southern blot showing absence of immunoglobulin gene rearrangements. Germline rearrangement JH, Cp., and CK gene regions in DNA from a control patient with reactive hyperplasia (C) and patient W. M. DNA was digested with EcoRI or BamHI and hybridized to JH, C~, and CK probes. Fragment sizes are shown in kilobases.
DISCUSSION
The karyotypic changes found in this patient were interesting for several reasons. First, the presence of c]onal abnormalities in 25 of 34 cells is consistent with a neoplastic process, although the diagnosis of lymphoma could not be established by histopathology. The patient died without development or regression of the disease. No germ-line karyotype was available, but the abnormalities observed are highly unlikely to be constitutional. Second, although a t(2;19) has not been previously noted in non-Hodgkin lymphoma, the breakpoints involved in this translocation are the site of several genes that are of interest in the study of oncogenesis. The bands 2pll-p12 include the sites of the kappa light chain [12] and the TGF-c~ locus [13]. Apart from frequent involvement in Burkitt's lymphoma, breaks at 2p11-p12 have been observed in at least nine cases of non-Burkitt, non-Hodgkin lymphoma, including diffuse, large cell ]ymphoma [15]. In addition, 19q13 is the site of the TGF-[3 gene [14], and breaks at this site have been reported in at least 17 cases of non-Hodgkin lymphoma, including nine with diffuse large cell lymphoma [15]. Third, although this particular rearrangement has not been reported, the breakpoints involved in the interstitial duplication of chromosome 2, 2q33 and 2q37, have been reported previously in two and five cases of non-Hodgkin lymphoma, respectively, especially diffuse large cell lymphoma [15]. These cytogenetic results, therefore, were suggestive of a neoplastic process. In order to determine if the patient had a B-cell tumor with detectable clonal immunoglobulin rearrangements, Southern blot analysis was performed with DNA from the second biopsy using probes for Cp., JH, CK, and C)~. This did not reveal any clonal immunoglobulin rearrangements. Similarly we found no evidence of a clonal rearrangement of the T-cell receptor genes using probes for TCR-~ and -~]. The sensitivity of our Southern blotting technique was such that a clonal rearrangement in at least 15% of cells could be detected. Approximately 74% of the cells we karyo-
256
G. Chenevix-Trench et al.
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23
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23
Figure 4 Southern blot analysis showing lack of T-cell receptor [3 and ~ chain gene rearrangement. DNA was digested with EcoRI and hybridized to T-cell receptor B and "y chain gene probes. Arrows indicate the position of germ-line bands in lane 1 (10.5 and 4.5 kb for TCR-[3 and 4,0 and 2.1 kb for TCR-~/); lane 2 shows rearrangements of [3 ~nd ~ genes in a Tcell acute lymphocytic leukemia patient as a positive control; lane 3 shows lack of rearrangements in [3 or ~ chain gene in DNA from patient W. M.
typed had chromosome abnormalities, thus, if these cells were clonal T or B cells, gene rearrangements should have been detected. The negative results obtained were surprising in light of the cytogenetic data. Such extensive cytogenetic aberrations would not be expected in a n o n m a l i g n a n t disorder, although few studies addressing this possibility have been published. Did the cytogenetic abnormalities occur in lymphoid stem cells prior to immunoglobulin or T-cell receptor gene rearrangements? The lack of such rearrangements would suggest an oncogenic event at an early stage of T- or B-cell development. This explanation is unlikely, however, because 82% of the lymphocytes expressed surface i m m u n o g l o b u l i n s and the remaining 18% of cells marked as T-lymphocytes. It is unlikely that the cytogenetic abnormalities observed were in metastasizing
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cells of the squamous cell carcinoma because metastasis of squamous carcinoma can be distinguished pathologically from atypical h y p e r p l a s i a and there was no evidence of such metastasis. Southern blot analysis d i d not reveal any rearrangement in the structure of the TGF genes. This could be because the w h o l e region has translocated without rearrangement of the BamHI or EcoRI sites. A similar event has been observed in acute myelocytic leukemia [16]. It is also worth noting that scanning of 30 kb of the cm y c gene in cases of Burkitt's l y m p h o m a k n o w n to have t(8;14) only revealed rearrangements of the c-myc locus in two of 11 cases [17]. Alternatively, while the translocation breakpoints and the TGF genes have been m a p p e d to a p p r o x i m a t e l y the same bands, they m a y be thousands of kilobases apart and, thus, uninvolved. In a d d i t i o n to our interest in the possibility that clonal rearrangements of the i m m u n o g l o b u l i n genes might be useful in the definitive diagnosis of a B-cell tumor, we were interested in the possibility that the k a p p a gene had been rearranged as a consequence of the t(2;19). However, Southern blot analysis d i d not provide any evidence for such a somatic rearrangement. The CK probe we u s e d hybridizes to only 13 kb of the k a p p a locus, thus, a translocation occurring outside this region w o u l d not be detected. Unfortunately there is no material available for in situ hybridization of any of these genes to further delineate the breakpoints of this interesting case. We propose that the cytogenetic abnormalities in this patient may have stimulated p o l y c l o n a l B-cell development. It was at this stage that the l y m p h nodes were b i o p s i e d and found to be expressing both k a p p a and l a m b d a i m m u n o g l o b u l i n chains. A clonal B-cell tumor might have arisen h a d the cells been subjected to a second "hit." A n analogy can be d r a w n w i t h African Burkitt's l y m p h o m a , in w h i c h it has been p r o p o s e d that p o l y c l o n a l B-cell activation results from chronic hypere n d e m i c malaria leading to e n h a n c e d p r e d i s p o s i t i o n to infection by Epstein-Barr virus [18]. The second " h i t " in Burkitt's l y m p h o m a is thought to be activation of the c-myc oncogene by c h r o m o s o m a l translocation. This w o u l d be in contrast to our proposal for the d e v e l o p m e n t of h y p e r p l a s i a in our patient, in w h i c h the polyclonal B-cell activation m a y have followed c h r o m o s o m a l mutations. However, it should be noted that the c h r o m o s o m e abnormalities observed in patient W. M., who was diagnosed as having atypical hyperplasia, differed from those reported in clones derived from a patient with clearly defined diffuse p o l y c l o n a l B-cell lymp h o m a [19]. Supported by NCI contract NCI-CP-21037 (J.M.C,U.F.). The TGF-ct and -6 probes were kindly provided by Dr. R. Derynck, Genentech Inc., South San Francisco, CA and the immunoglobulin probes by Dr. S. Korsmeyer. This article is #295 from the Department of Human Genetics, Richmond, VA.
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