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For personal use only. 1994 84: 3648-3652 Diagnosis of t(2;5)(p23;q35)-associated Ki-1 lymphoma with immunohistochemistry M Shiota, J Fujimoto, M Takenaga, H Satoh, R Ichinohasama, M Abe, M Nakano, T Yamamoto and S Mori Updated information and services can be found at: http://www.bloodjournal.org/content/84/11/3648.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on November 17, 2014. For personal use only. RAPID COMMUNICATION Diagnosis of t(2; 5)(p23; q35)-Associated Ki-l Lymphoma With Immunohistochemistry By Mami Shiota, Jiro Fujimoto, Masato Takenaga, Hitoshi Satoh, Ryo Ichinohasama, Masafumi Abe, Masaru Nakano, Tadashi Yamamoto, and Shigeo Mori Some Ki-l lymphomas carry a specific chromosomal translo- ent on biopsied lymphomas, reverse transcriptass-polymercation, t(2;51(p23;q35). W e have recently found a novel hyas8 chain reaction (RT-PCR) covering the fusion junction of perphosphorylated 80-kD protein tyrosine kinase, p80, in a p80 mRNA was performed. Among 10 Ki-l lymphomas and human Ki-l lymphoma with this translocation. Subsequent l 0 additional lymphomas other than the Ki-l lymphomas, cDNA cloning showed that p80 is a fusion protein of two expression of p80 mRNA was detectedin three cases excludifferent genes on chromosome 2p23 and 5q35, the novel sively. When these 20 cases and additional 30 lymphomas tyrosine kinase gene and nucleophosmin gene, respectively.were immunostained with anti-p80, positive staining was In this study, we intended to detect p80 on lymphoma tisnoted exclusively in the three cases found by PCR to have sues with immunologic methods. Thus,we developed rabbit harbored the p80 mRNA. Thus,the present immunostaining, as well as PCR, wasshown to beefficientfordetecting polyclonal antibody using a synthetic peptide corresponding to a part of its kinase domain. The antibody (anti-p80) immu-lymphomas producing this chimeric protein/mRNA. 0 1994 by The American Societyof Hematology. noprecipitatedandimmunoblottedp80specificallyfrom AMS3. Then, to examine whether t{2;5)(p23;q35)was pres- K 1-1 LYMPHOMA, also called anaplastic large cell lymphoma or large cell anaplastic lymphoma, is a subtype of human non-Hodgkin's lymphoma (NHL) characterized by expression of CD30 (Ki-l antigen) and its peculiar large neoplastic cells, which mimick HodgkidReed-Sternberg cells.'.* Recent studies have shown that Ki-l lymphomas comprise a heterogeneous group, differing in their clinicalcourse,histology,immunohistology,and Around one third of all Ki-l lymphomas carry a chromosomal translocation, t(2;5)(~23;q35),"~and these cases are suggested to constitute a unique subgroup sharing an identical genetic background. However, the clinical and biological specificity of cases with t(2; 5 ) have not yet been elucidated. We recently established a Ki-l lymphoma cell line with t(2;5) inmicewith severe combined immunodeficiency (SCID)." Subsequent analysis of signal transduction in this cell line showed hyperphosphorylation of a unique, phosphotyrosine-containing protein of Mr 80,000, designated p80. Amino acid sequence analysis of tryptic digests showed that p80 is a novel protein tyrosine kinase similar to LTK." Subsequent cloning of p80 cDNA showed that the p80 gene is a fusion gene made up partly of a novel protein tyrosine kinase and partly of nucleophosmin (unpublished observations, October 1994). As characterization of this cDNA was being completed, the same cDNA was identified by posiFrom The Departments of Pathology and Oncology, The Institute of Medical Science, The University of Tokyo; and Nichirei, CO, Tokyo, Japan. Submitted August 19, 1994; accepted September 26, 1994. Supported in part by the Ministry of Education, Science, and Culrure, Japan (grant no. 04557019). Address reprint requests to Mami Shiota, MD, Department of Pathology, TheInstitute of Medical Science, The University of Tokyo, Tokyo 108, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8411-0045$3.00/0 3648 tional cloning of this chromosomal breakpoint by another group." Thus, it is now evident that p80 is a chimeric protein that characterizes Ki- 1 lymphomas bearing t(2; 5)(p23;q35). These results promoted us to study Ki- l lymphomas using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistology, anticipating that the results would be of value for diagnosis of this specific subtype, as well as the study of p80-mediated intracellular events. MATERIALSAND METHODS Cells and tissues. A human Ki-l lymphoma cell line AMS3, bearing the reciprocal chromosomal translocation t(2;5)(p23;q35), was maintained in SCID mice, The details of AMS3 have been described previously." SurgicalIy excised and fresh-frozen human tissues, comprising three nontumorous tonsils and 50 malignant lymphomas, were selected from our tissue library. The lymphomas comprised 10 Ki-l lymphomas, 6 HLs, and 24 NHLs. The 24 NHLs consisted of 6 T-cell NHLs including 3 diffuse large noncleaved cell type, 2 immunoblastic type, and l lymphoblastic type; 16 Bcell NHLs including 2 follicular lymphoma, 3 diffuse small lymphocytic type, 9 diffuse large noncleaved type, and 2 immunoblastic type; and 2 non T-non B cell NHL with the histology of immunoblastic type. Three of them were known to ear t(2;5)(p23;q35), whereas cytogenetic data were not available for most of the other cases. The specimens had been embedded in OCT compound (Miles Laboratories, Kankakee, IL), snap-frozen in n-hexane precooled with dry ice-acetone and stored at -70°C until use. All of these cases were used for immunohistologic study, and 20 rather well-presereved cases were also used for RT-PCR assay. The paraffin-embedded sections of some of these cases were also used for immunohistology. In vitro-maintained human T-cell leukemia cell lines, Jurkat and MT- 1, were used for Western blotting. None bore the t(2;5) chromosomal translocation. The cells were cultured in RPM1 1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 10% (voVvol)fetal bovine serum (ICN Biochemical, Tokyo, Japan) at 37°C and 5% COZ.Peripheral blood mononuclear cells (PBMC) isolated by centrifugation with Ficoll-Hypaque (Pharmacia LKB Biotechnology, Uppsala, Sweden) from healthy donors were also used as another control. RT-PCR. RT-PCR was performed to clarify the presence of p80 fusion mRNAs in stained lymphomas. RNAs were isolated from 20 lymphomas, comprising 10 Ki-l lymphomas, 6 T-cell lymphomas, 4 B-cell lymphomas, and also I nontumorous tonsil, using the guanidinium-acidphenol extraction method." To make cDNA pools, Blood, Vol 84, No 11 (December I). 1994: pp 3648-3652 From www.bloodjournal.org by guest on November 17, 2014. For personal use only. 3649 p80 EXPRESSION IN Ki-l LYMPHOMA total RNAs (10 pg) were reverse-transcribed with 100 U of modified RT from Moloney Murine Leukemia Virus (Superscript; BXL) in a total volume of 25 pL according tothe manufacturer’s protocol Each cDNA pool (0.5 pL) was PCR-amplified in 50 pL of solution containing 10 mmol/L Tris (pH 8.3), 50 mmoVL KC1, 1.5 mmoVL MgCl,, 200 p m o K d N T P s , 30 pm01 of primers, and 2.5 U of Taq polymerase (Toyobo, Japan). Three primers were prepared to detect two cDNAs, those of nucleophosmin for nonfusion gene and p80 for fusion gene. Thus, the forward primer for both reactions was S’-GGCAGTCCAA’ITAAAGTAACAC-3’, the reverse primer for nucleophosmin cDNA (M23613), 5’-TGGAACC”TGCTACCACCTC-3‘, and for p80, 5’-GAGCTTGCTCAGCTGTACTC-3’. With the use of these primers, a 252-bp product was expected for nucleophosmin cDNA, and a 225-bp product for p80 cDNA. The reaction was performed for 2 minutes at 94°C for 1 cycle, 30 seconds at 9 4 T , 30 seconds at W C , and 2 minutes at 72°C for 40 cycles. Antibody againsrp80 (mti-p80). The antibody against p80 (antip80) was raised by immunizing rabbits with a synthetic peptide SNQEVLEFVTSGGR, the sequence obtained in our previous study,’’ corresponding to the putative kinase domain. The specific antibody was purified by affinity chromatography on columns to which the synthetic peptide had been linked covalently. Western bloning. AMs3 cells were lysed with RIPA buffer (1% vol/vol, Triton X-100) and the cleared lysates were immunoprecipitated with anti-p80. The whole lysates and the immunoprecipitates were then subjected to sodium dodecyl sulfate (SDS)B% polyacrylamide gel electrophoresis (PAGE) under reducing conditions and then transferred to polyvinylidene difluoride membrane (Bio-Rad, CA), which was subsequently blocked by incubation with bovine serum albumin (50 mg/mL). The blot was probed in turn with the anti-p80 antibody (1 mg/mL), diluted 1:1,O00, and the preimmune serum and the antibody preabsorbed with excess synthetic peptide used as the antigen. Irnrnunohisrochernical study. The labeled avidin-biotin (LAB) method was used to demonstrate p80 and other antigens on tissue section^.'^ To detect p80 and some other lymphocyte markers, antip80 and several CD antibodies described in the Results section were used as the first immunohistology reagents. The preimmune serum, previously taken from the rabbit that harbored anti-p80 after immunization, was also used as a control serum. For the second- and thirdphase reagents, biotinylated antirabbit Ig (E353; DAKOPA’ITS, Denmark) and streptavidin (E364; DAKOPAITS) were used. The staining procedures were as follows: Frozen sections (5-pm thick) were cut with a cryostat and fixed with periodate-lysine paraformaldehyde (PLP) solution for 15 minutes. They were then washed with phosphate-buffered saline (PBS), incubated with appropriately diluted antibody for 1 hour, washed with PBS, incubated with the second- and third-phase reagents for 30 minutes, and washed. They were then stained with 0.6 mg/mL 3.3’-diaminobenzidine tetrahydrochloride (Sigma Laboratories CO, St Louis, MO) in PBS containing 0.01% HZOZ for 5 minutes. The immunostaining of paraffin sections was done by the method of Shi et al.’5.’6 RESULTS RT-PCR analysis was performed to show the expression of the fusion gene. Although 252-bp products corresponding to the control nucleophosmin cDNA were found in all 21 samples including 20 NHLs and 1 normal tonsil, the 225bp products corresponding to the p80 cDNA were found exclusively in 3 cases (Fig 1). These 3 cases were found to be the same ones found in the previous analysis to bear the chromosomal translocation t(2; 5)(p23;q35), suggesting that this RT-PCR detected this chromosomal translocation spe- cifically. All 3 of these cases were Ki-l lymphomas and not other subtypes. On Western blotting, anti-p80 demonstrated an 80-kD band on AMs3 but not on the other cell lines, Jurkat and MT-1, or on PBMC from a healthy donor. No extrabands were observed in any of these cells. The 80-kD band was eliminated by preabsorbing the 1:1,000-diluted antibody with excess synthetic peptide that was used as the immunogen (Fig 2). On immunostaining with anti-p80, most of the AMs3 cells were heavily stained, the reaction being evident in the nuclei and cytoplasm. This immune reaction was observed up to a dilution of 1:13,500 (Fig 3). Meanwhile, in the control tonsil tissues, a weak reaction was noted on follicular dendritic cells and endothelial cells. However, this reaction was very faint and easy to differentiate from the heavy reaction of AMS3. In addition, such reactions were not observed in any of the lymphoma tissues. AMs3 wasnot stained by the 1:1,000-diluted preimmune serum. Among the 50 lymphomas that were subjected to immunostaining, only three reacted with anti-p80. All of these three cases were the Ki-l lymphomas that had been shown in the preceding cytogenetic study to bear t(2;5)(p23;q35), and also those that expressed p80 mRNA detected by RT-PCR. The staining of these three specimens was clear and definite, the reaction being found in most, if not all, of the neoplastic large cells. However, the intracellular locations of the immune reaction differed among the cases. Thus, in case 1, nuclei were most heavily stained, and definite nuclear staining was observed in about two thirds of the neoplastic cells of this particular case. In cases 2 and 3, the cytoplasm was most heavily stained, whereas nuclei were stained occasionally in about one third of the neoplastic cells. The cell membrane did not seem to be the main locus of immunostaining. The paraffin sections of AMs3 also reacted with this antibody, clearly and at similar loci (Fig 4). On hematoxylin-eosin staining, all of the 10 Ki-l lymphomas showed a similar anaplastic large cell morphology, and no clear differences were noted between anti-p80-stained and unstained cases. Also, on immunostaining with CD3 (Becton Dickinson [BD] CA), CD4 (BD), CD8 (BD), CD15 (BD), CD20 (Dakopatts, Denmark), CD25 (BD), CD71 (Nichirei, Tokyo, Japan) and antiepithelial membrane antigen (EMA, Dakopatts), no differences were noted between p80-positive and -negative eases. DISCUSSION PCR and RT-PCR have been used to detect chromosomal translocations in hematologic and other malignancies.” They are especially useful for revealing the discrete loci of genomic DNA or cDNA containing the fusion junction. In the present study, t(2;5)(p23;q35) was advantageous for RTPCR study because the cDNA sequences around the fusion junction were identical in all five cases studied thus far, including our AMS3 cells.11,12 The primer pairs used in the present RT-PCR were prepared to show this fusion junction, and 3 of 20 cases were found to harbor 225-bp products of the expected size. As expected, these three cases were the same Ki-l lymphomas that, on previous cytogenetic study, From www.bloodjournal.org by guest on November 17, 2014. For personal use only. SHIOTA ET AL 3650 L 1 2 3 4 5 6 7 8 9 10 11 12 13 1 4 15 16 l7 18 19 20 21 22 Fig 1. Demonstration oft(2;51(p23;q35) by RT-PCR. The 225-bp product indicates thepresence of p80 chimeric mRNA from the fusiongene junction, whereas the 252-bp product corresponds t o nucleophosmin mRNA from nontranslocated chromosome 5q35. The p80 cDNA was demonstrated in cases 1 through 3 exclusively. (Lane 21,D-H,O as a negative control.) had been shown to carry t(2;5)(p23;q35). The succeeding immunohistologic study using anti-p80 also showed that these three cases, and none of the other 47 cases, reacted with this antibody. Thus, the results of RT-PCR and immunostaining corresponded perfectly. The immune reaction on t(2;5)-carrying Ki-l lymphomas was found in both cytoplasm and nuclei, although the ratio of stained nuclei varied among the cases. Also, the intensity of nuclear or cytoplasmic staining vaned among neoplastic cells in the same tissue section. These variations in immunostaining may somehow be related to the molecular function of p80 in these cells. Wang et allxreported that nucleophosmin was predominantly found in nucleoli, and partly in cytoplasm fractions, presumably associating with cytoskeletal elements. Present results in terms of localization of p80 may somehow be related to the intracellular localization of nucleophosmin.'* In the meantime, it is necessary to be cautious about the S 1 2 specificity of anti-p80. Anti-p80 was thought initially to recognize p80 specifically. Thus, an oligopeptide corresponding to a unique part of the kinase domain was selected as the immunogen. Later, upon cloning of p80 cDNA, as well as upon recently published sequences of cDNA covering the junction of t(2;5),'* this oligopeptide was confirmed to be encoded by the kinase portion of unique tyrosine-kinase, termed as ALKby Moms et al.'* Therefore, wehad to consider the possibility that anti-p80 may also react with kinase portion ALK. However, on immunostaining and Western blotting, no lymphomas or peripheral blood lymphocytes not carrying t(2;5) gave positive signals, suggesting that anti-p80 can be used on human lymphomas and non-neoplastic lymphocytes to detect p80 specifically. LTK is another protein tyrosine kinase that may crossreact with anti-p80, because its catalytic domain is highly homologous to p80. Thus, we immunostained a human erythroid leukemia cell line K562, which is known to express LTK." S Fig 2. Anti-p80 shows an 80-kD band corresponding t o p80 in the Ki-l lymphoma cell line AMS3, but not in control cells. Cell l y s a t e s were immunoprecipitated with 25.2G4 (anti-phosphotyrosine antibody; Wako Biochemicals, Japan) and the immunoprecipitates were further immunoblotted withantbp80 (lane 1).Whole lysates of AMs3 (lane 2). the controlcell lines MT1(lane 4) and Jurkat(lane 5).and mononuclear cells from a healthy control (lane 6) were directly immunoblotted with anti-p80. Lane 3, AMs3 cell lysates immunoblotted with anti-p80 preabsorbed with an excess of the syntheticpeptide. Lane S, Rainbow proteinmolecular weight markers (Amersham, Japan). p80 is detected only in lanes 1 and 2. From www.bloodjournal.org by guest on November 17, 2014. For personal use only. p80 EXPRESSION IN Ki-l LYMPHOMA 3651 Fig 3. lmmunostaining of four biopsied fresh-frozen tissues of Ki-l lymphomas. (a) lmmunostaining with CD30 (DAKO-BerH2, DAKO). (b) lmmunostaining with anti-p80. (c) Hematoxylin-eosin staining. Cases 1,2, and 3, but notcase 4, are known to bear the chromosomaltranslocation t(2;51(p23;q35). Clear immunostaining isobserved in cases 1 through 3, but notin case 4, whereas all fourcases react strongly with CD30. In case 1, nuclei are stained in about t w o thirds of the neoplastic cells, whereas in cases 2 and 3 the cytoplasm isstained. of Fig 4. lmmunostaining paraffin section of case 1 with anti-p80. Clear immune reaction, equivalent t o Fig 3, is noted. As the result, no staining was noted on K562, whereas heavy staining was found on A M S 3 , the p80-carrying Ki-l lymphoma cell line, apparently suggesting the absence of cross-reaction between anti-p80 and LTK (data not shown). Also, LTK is known to be expressed on normal immature and mature B cells of miceZoas well as human (Hirai H., personal communication, October 1994). Our immunostaining of normal human tonsils or B-cell NHLs did not show any positive reaction with non-neoplastic or neoplastic B cells on these tissues, again suggesting the absence of cross- reactivity of this antibody with LTK. Meanwhile, the reaction of follicular dendritic cells observed in non-neoplastic tonsil must be studied further in detail to show their antigenic specificities, even though these reactions were far weaker and not evident in nuclei, and thus easy to differentiate from reactions with t(2;5)-associated lymphomas. The breakpoints of the chromosome translocation are often dispersed in certain restricted introns, leading to the production of, occasionally, a variety of different chimeric proteins." However, there remains a less likely possibility in case of t(2;5)(p23;q35) that there exists another type of translocation that does not produce p80 and is not detected by the present RT-PCR. As yet, we cannot rule out this possibility because a cytogenetic study was not done on a large proportion of the present RT-PCR-negative or p80negative cases. However, this variant, if present, will remain in a very minor group, because all the previously reported or present cases with t(2;5)(p23;q35) studied so far showed identical cDNA sequences around the fusion junction. Ki-l lymphoma is known to constitute a heterogeneous It is evident that cases with t(2;S) should be differentiated from other Ki- 1 lymphomas because they apparently From www.bloodjournal.org by guest on November 17, 2014. For personal use only. 3652 SHIOTA ET AL differ in their genetic background. The demonstration of p80 will contributemuchtothisdifferentiation.Our anti-p80 antibodyhas aspecificbenefitin thatitcanbeusedon paraffin sections. The application of this antibody to a wide range of paraffin-embedded Ki-l lymphomas will show the identity of the t(2;5) subgroup in Ki- 1 lymphomas in terms of their clinical course, histology, and other phenotypic characteristics. REFERENCES 1. Stein H, Mason DY, Gerdes J, O’Conner N, Wainscoat J, Pallesen G , Gatter K, Falini B, Delsol G, Lemke H, Schwarting R, Lennert K: The expression of the Hodgkin’s disease associated antigen Ki-l in reactive and neoplastic lymphoid tissue: Evidence that Reed-Stemberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood 662348, 1985 2. 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