970 Hodgkin Disease with Subsequent Transformation to CD30 Positive Non-Hodgkin Lymphoma in Six Patients Naoyoshi Mori1 Kazuko Watanabe1 Yoriko Yamashita1 Atsuo Nakayama1 Yasushi Yatabe2 Michihiko Narita3 Toshitaka Kobayashi4 Yuuichi Hasegawa5 Hiroshi Kojima5 Toshiro Nagasawa5 1 First Department of Pathology, Nagoya University School of Medicine, Nagoya, Japan. 2 Department of Pathology and Clinical Laboratories, Aichi Cancer Center Hospital, Nagoya, Japan. 3 Department of Pathology, Koseiren Kamo Hospital, Toyota, Japan. 4 Department of Internal Medicine, Tsuchiura Kyodo Hospital, Tsuchiura, Japan. 5 Department of Internal Medicine, Tsukuba University, Tsukuba, Japan. Supported in part by a grant-in-aid from the Ministry of Education (09670183) The authors thank Mr. Nobuaki Misawa and Yoshie Ieda for their excellent technical assistance. Address for reprints: Dr. Naoyoshi Mori, Department of Pathology, Nagoya University School of Medicine, Tsuruma-cho 65, Nagoya, showa-ku, Nagoya, 466-8550, Japan. Received January 9, 1998; revision received April 13, 1998; accepted July 31, 1998. © 1999 American Cancer Society BACKGROUND. Previous studies have indicated that some patients with Hodgkin disease have an aggressive clinical course. However, their characteristics have not been elucidated. METHODS. Six patients initially diagnosed as having Hodgkin disease with subsequent transformation to CD30 positive non-Hodgkin lymphoma were clinically and immunohistochemically studied. Ten patients with classic anaplastic large cell lymphoma (ALCL) and 19 patients with classic Hodgkin disease were studied for comparison by immunohistochemistry. RESULTS. Five patients, excluding one whom the authors recently encountered, had an aggressive clinical course and died within approximately 1 year after diagnosis; four of them were autopsied. Common histologic findings in the six patients included sheets of atypical mononuclear cells and sinusoidal infiltration of small numbers of these cells, as well as Reed–Sternberg cells or lacunar cells. Follow-up biopsies revealed histologic progression to ALCL in which sheets of atypical mononuclear or multinucleated cells spread throughout the lymph nodes. Immunohistochemically, epithelial membrane antigen, granzyme B, perforin, and pancadherin were expressed frequently in the six patients and in ALCL but were not expressed in Hodgkin disease. Latent membrane protein-1 (LMP-1) and Epstein–Barr virus-encoded RNA (EBER-1) were totally negative in the six patients and in ALCL but were expressed frequently in Hodgkin disease. In contrast, LeuM1 (CD15) was expressed frequently in the six patients and in Hodgkin disease, whereas it was not expressed in ALCL. MT1 (CD43), UCHL-1 (CD45RO), and P80 were totally negative in the six patients and in Hodgkin disease, whereas they were expressed variably in ALCL. CONCLUSIONS. These results suggest that the six patients studied had intermediate phenotypes between ALCL and Hodgkin disease. The authors also suggest that patients with sheets of atypical mononuclear cells and sinusoidal infiltration of atypical mononuclear and multinucleated cells have an aggressive clinical course and should be treated with intensive chemotherapy. Cancer 1999;85:970 –9. © 1999 American Cancer Society. KEYWORDS: Hodgkin disease, anaplastic large cell lymphoma, granzyme B, perforin, Epstein–Barr virus-encoded RNA, latent membrane protein-1 (LMP-1), pancadherin, p80. R ecent studies have revealed that Hodgkin disease and anaplastic large cell lymphoma (ALCL)1–5 have various features, such as the presence of t(2;5)(p23;q35) translocation6 –14 in ALCL and its absence of Hodgkin disease. Furthermore, ALCL may have null-cell or T-cell phenotypes, whereas Hodgkin disease occasionally has B-cell phenotypes.15–18 However, there are some patients in which the disease is quite difficult to differentiate.19,20 In addition, some reports indicate that some patients with Hodgkin disease progressed to ALCL.21,22 Transformation of Hodgkin Disease to ALCL/Mori et al. 971 TABLE 1 Histologic and Immunohistochemical Characteristics of the Initial Lymph Node Biopsy Specimens from the Six Patients Patient Characteristics 1 2 3 4 5 6 Histologic subtype Fibrosis Sheets of atypical cells Sinus involvement RS cells Lacunar cells Main immunophenotypes BerH2 LeuM1 EMA LCA L26 CD3 LD 2 111 11 1 2 LD 2 111 1 1 2 MC 2 111 1 1 2 MC 2 11 1 1 2 NS syn 11 11 1 1 1 NS syn 11 11 1 1 11 11 1 1 2 2 2 11 2 2 2 2 2 11 1 2 2 2 2 1 11 2 2 2 2 11 11 1 2 2 2 1 11 2 2 2 2 LD: Hodgkin disease, lymphocytic depletion; MC: Hodgkin disease, mixed cellularity; NS syn: Hodgkin disease, nodular sclerosis, syncytial variant; RS cells: Reed-Sternberg cells; EMA: epithelial membrane antigen; LCA: leukocyte common antigen; 2: negative; 1: positive; 11: strongly positive. Based on these results, the Revised European and American Lymphoma (REAL) classification23 has proposed a provisional entity of Hodgkin-like ALCL.24 However, this entity has ambiguous features along with its morphology. The authors had experience with six patients who were diagnosed with Hodgkin disease on initial biopsies but who had subsequent biopsies that revealed ALCL and had an aggressive clinical course. Further studies of such patients may elucidate the correlation between Hodgkin disease and ALCL. MATERIALS AND METHODS Paraffin Tissue Lymph node samples were obtained, fixed in a 10% formaldehyde solution, and embedded in paraffin. Sections were cut 2– 4 mm thick and were stained with hematoxylin and eosin. An avidin-biotin-peroxidase complex (Nichirei Corp., Tokyo, Japan) method25 was used for all immunohistochemical studies [BerH2 (CD30), LCA (CD45), epithelial membrane antigen (EMA), L26 (CD20), polyclonal CD3, CD8, and LMP-1 from DAKO (Copenhagen, Denmark); UCHL-1 (CD45RO) from Nichirei Corp.; LeuM1 (CD15) from Beckton Dickinson Monoclonal Center (Mountain View, CA); MT1 (CD43) from Bioscience (Emmerbruecke, Switzerland); granzyme B (GrzB) from Monozan (Ueden, Netherlands); pancadherin (p-cadherin) from Sigma Biosciences (St. Louis, MO); and p80NPM/ALK from Nichirei Corp.]. Frozen Tissue Fresh tissue samples were first fixed in periodatelysine-paraformaldehyde,26 frozen, sectioned 6-8 m thick with a cryostat, postfixed in acetone for 10 minutes, and then incubated with the primary antibodies [Leu1 (CD5), Leu2 (CD8), Leu3 (CD4), Leu4 (CD3), Leu19 (CD56), and Ki-1 (CD30), from DAKO; B1 (CD20) and B4 (CD19) from Coulter Immunology (Hialeah, FL); bF1 and TCRd1 from T Cell Sciences (Cambridge, MA); and perforin from T Cell Diagnostics, Inc. (Cambridge, MA)]. The avidin-biotin-peroxidase complex method25 was also used. In Situ Hybridization After deparaffinization and digestion with proteinase K (Sigma Chemical Co., St. Louis, MO), the tissue sections were hybridized with fluorescein-conjugated oligonucleotide probes for Epstein–Barr virus (EBV)encoded RNA (EBER-1; 30 bp; EBV probe in situ hybridization kit; Novocastra Laboratories Ltd., Newcastle, United Kingdom). Stringently washed sections were reacted with an antifluorescein antibody, and the reactions were visualized with 5-bromo-4-chloroindolylphosphatase and nitroblue tetrazolium salt. Details of the in situ hybridization procedure have been described elsewhere.27–31 Southern Blot Hybridization Ten micrograms of high-molecular-weight DNA extracted from each sample were digested with EcoRI, BamHI, and HindIII (Nippon-Gene Corp., Tokyo, Japan) restriction enzymes. Electrophoresis of the resulting fragments was performed by using 0.7% agarose gel slabs, followed by Southern blotting32 with JH, CTb, and Jr probes labeled with 32phosphorus. After 972 CANCER February 15, 1999 / Volume 85 / Number 4 FIGURE 1. Hematoxylin and eosin (H&E) staining of an initial lymph node biopsy reveals a few Reed–Sternberg cells with prominent nucleoli (patient 4; 3600). appropriate washing, autoradiography was performed at 280°C for 4 days. RESULTS All of the patients were diagnosed with Hodgkin disease on initial biopsies but then underwent a rapid clinical course with the exception of one patient. The histologic characteristics of the six patients are shown in Table 1. Of these, four patients were characterized by scant fibrosis, revealing histologic features of mixed cellularity (Fig. 1) or of a lymphocyte-depleted type of Hodgkin disease. In these patients, typical, isolated Reed–Sternberg cells with large cytoplasms and eosinophilic, prominent nucleoli were observed surrounded by nonneoplastic lymphocytes in the entire lymph nodes. In addition, these lymph nodes also contained small clusters of atypical mononuclear or multinucleated cells (Fig. 2) and small numbers of these cells in sinuses (Fig. 3). An admixed inflammatory cell infiltrate composed of eosinophils and epithelioid cells was variably noted. The remaining two FIGURE 2. H&E staining of an initial lymph node biopsy reveals sheets of atypical mononuclear and multinucleated cells (patient 2; 3300). patients, who were considered to have Hodgkin disease, nodular syncytial variant, had biopsies that were characterized by thick, fibrous connective tissue that encircled lymphoid tissue, which contained Reed– Sternberg cells and lacunar cells (Fig. 4), as well as sheets of atypical mononuclear cells. Plasma cells, neutrophils, eosinophils, and necrosis were also observed. Main immunophenotypic findings of the initial lymph node biopsies from the six patients showed that Reed–Sternberg cells or their variants were positive for BerH2 in 6 of 6 patients, for LeuM1 (Fig. 5) in 5 of 6, for EMA in 2 of 6, for LCA in 1 of 6, for L26 in 0 of 6, and for CD3 in 0 of 6 (Table 1). Based on these histologic and immunohistochemical findings, we diagnosed these patients with Hodgkin disease on initial biopsies. All of the patients were treated after initial diagnosis (Table 2). Of these, one patient (6) responded well to chemotherapy, and complete remission was achieved. Two patients (1 and 2) responded relatively well to chemotherapy, with significant decrease of lymphadenopathy. However, lymph node enlarge- Transformation of Hodgkin Disease to ALCL/Mori et al. FIGURE 3. H&E staining of an initial lymph node biopsy reveals sinusoidal infiltration of a small number of atypical mononuclear cells (patient 1; 3600). ment recurred shortly after chemotherapy, and the patients underwent a progressive clinical course with systemic lymphadenopathy and bone marrow involvement. The remaining three patients responded poorly to chemotherapy, with partial reduction of lymphadenopathy, and underwent an aggressive clinical course. Follow-up biopsies were obtained from patients with recurrent lymph nodes after successful treatment or after partial response to chemotherapy. Follow-up biopsies revealed histologic progression to ALCL in which sheets of atypical mononuclear or multinucleated cells spread throughout the lymph nodes. In addition, biopsies of the skin, liver, spleen, and bone marrow often revealed lymphomatous involvement in many of the patients. Subsequent biopsies of various organs and tissues did not show significant change of the phenotypes of neoplastic cells (Table 3). However, some of the neoplastic cells became positive for EMA in two additional patients (2 and 3). Ten patients with classic ALCL and 19 patients with Hodgkin disease were studied for comparison by immunohistochemistry (Table 4). Cytotoxic mole- 973 cules, GrzB (Fig. 6) and perforin, were expressed in 4 of 6 patients. Either one or both of the cytotoxic molecules were expressed in 8 of 10 patients with classic ALCL. In contrast, only one of the patients with Hodgkin disease expressed perforin. Pan-cadherin was expressed in all our patients (Fig. 7) and in patients with classic ALCL but was expressed only in 10 of 19 patients with Hodgkin disease. Latent membrane protein-1 and EBER-1 were not expressed in any of the current patients or in patients with classic ALCL. Latent membrane protein-1 and EBER-1 were expressed in 11 patients and 13 patients, respectively, with Hodgkin disease. MT1 (CD43) and UCHL1 (CD45RO) staining were absent both in our patients and in those with Hodgkin disease but were identified in 6 patients and 5 patients, respectively, with classic ALCL. Leu19 (CD56) was positive in two samples from our patients and in three samples from patients with classic ALCL. P80NPM/ALK was totally negative in our patients and in patients with Hodgkin disease but was positive in 7 patients with classic ALCL. Southern blot hybridization studies revealed that none of the patients studied possessed a clonal rearrangement band of T-cell receptor b chain (Cb1). All but one patient died within approximately 1 year after diagnosis. Autopsies performed on four patients revealed lymphomatous infiltration of various organs. The involved organs contained lymphomatous cells that tended to form clusters (Fig. 8). Variable fibrosis was noted, and occasional nodules larger than 1 cm in greatest dimension were formed. Skin involvement was observed in four patients, liver involvement was observed in four patients, lung involvement was observed in four patients, spleen involvement was observed in three patients, and bone marrow involvement was observed in two patients. Systemic lymph node involvement was seen in all patients. DISCUSSION Strickler et al.33 reported on 18 patients with Hodgkin disease with sheets and cohesive clusters of numerous Reed–Sternberg cell variants and called them a syncytial variant of nodular sclerosing (NS) Hodgkin disease. MacLennan et al.34 studied 1659 patients who were treated for Hodgkin disease and found that NS Hodgkin disease with extensive lymphocyte depletion or numerous anaplastic Hodgkin cells (Grade II NS) is resistant to initial therapy, frequently recurs, and has a worse prognosis than Grade I NS. They concluded that patients with Grade II NS require more aggressive initial therapy. This work formed the basis for the ALCL Hodgkin-like subtype in the REAL classification.23 However, a great deal remains to be elucidated about the nature of Hodgkin-like ALCL. Two of our 974 CANCER February 15, 1999 / Volume 85 / Number 4 FIGURE 4. (a) H&E staining of an initial lymph node biopsy reveals thick, fibrous connective tissue encircling lymphoid nodules (patient 5; 330). (b) Higher magnification reveals numerous lacunar cells (3300). patients were consistent with Grade II NS, and the remaining four patients were considered to have the mixed cellularity or lymphocyte depleted types on initial biopsies. We studied six patients retrospectively who were diagnosed initially with Hodgkin disease, but their subsequent biopsies revealed ALCL features, and their disease underwent a rapid, aggressive clinical course. This is the first precise report to reveal the histologic progression of Hodgkin disease to ALCL in sequential biopsies. Some previous reports have indicated a close correlation between Hodgkin disease and nonHodgkin lymphoma. Sundeen et al.35 reported lymphocyte predominant Hodgkin disease, nodular subtype, with coexistent large cell lymphoma. Others have reported that some patients had Hodgkin disease that progressed to non-Hodgkin lymphoma, including ALCL,21,22 and some patients had ALCL that changed into Hodgkin disease.20 Our patients belong to the second type. These patients with secondary ALCL reportedly have worse prognoses than those with primary ALCL.36 Pileri et al.22 reported on five patients with Hodgkin disease associated with ALCL and suggest that ALCL lymphoma with previous or simultaneous Hodgkin disease should be regarded as the blastic transformation of Hodgkin disease because of the occurrence of both neoplasms in one patient and the onset of ALCL lymphoma in the same anatomical sites as those of Hodgkin disease and because of the short interval between Hodgkin disease and ALCL in three of five of their patients. Those authors also argued in favor of the blastic transformation of previous Hodgkin disease by the absence of any significant variation in the phenotype of the neoplastic cells at presentation and at the time of second biopsy. Our current study also had similar results. Immunophenotypic studies of our patients did not reveal any significant change in the phenotypes of the neoplastic cells between the initial biopsies and subsequent biopsies. Our patients showed the morphology of Hodgkin disease on initial biopsy with foci of ALCL that progressed subsequently to ALCL with an aggressive clinical course and was accompanied frequently by early skin, liver, spleen, bone marrow, and systemic lymph node involvement. Transformation of Hodgkin Disease to ALCL/Mori et al. 975 TABLE 2 Clinical and Autopsy Findings from the Six Patients Patient Sex/age (yrs) Involved sites clinical (biopsies) Stage Therapy Clinical course Involved sites at autopsy 1 M/45 Cervical,a aortic, inguinal LN,a spleen, skin,a bone marrowa IIIB C-MOPP 7.9 mo (dead) 2 M/31 Mediastinal,a paraaortic LN, bone marrow,a livera IVB C-MOPP 6.8 mo (dead) 3 4 M/69 F/38 Cervical,a aortic, inguinal LN, skina Cervical,a paraaortic LN,a bone marrow,a spleen,a skina IVB IVB COP C-MOPP 7.8 mo (dead) 13 mo (dead) 5 M/77 IVB C-MOPP 11 mo (dead) 6 M/23 Cervical,a mediastinal, paraaortic,a LN, liver, spleen, lung, skina Cervical,a mediastinal,a supraclavicular LNa Liver, spleen, bone marrow, skin, lung, pericardium, kidneys, lymph nodes Liver, spleen, bone marrow, lung adrenals, dura mater, intestines, ureters, lymph nodes — Liver, bone marrow, lung, stomach pharynx, lymph nodes Liver, spleen, skin, lung, lymph nodes IIB CDDP 1 CHOP 5 mo (CR) — M: male; F: female; MOPP: mechlorethamine, vincristine, procarbazine, and prednisone; COP: cyclophosphamide, vincristine, and prednisone; CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone; CDDP: cisplatin; CR: complete remission; mo: month; LN: lymph node. a Biopsied tissues or organs. TABLE 3 Immunohistochemical and Immunogenetic Findings of Lymph Node Biopsies with Anaplastic Large Cell Lymphoma Features from the Six Patients Patient FIGURE 5. LeuM1 staining of an initial lymph node biopsy reveals positive reactivity in many atypical cells (Patient 4) (3300). Initial biopsies from all of our patients contained small numbers of atypical BerH2 (or LeuM1) positive mononuclear, or multinucleated atypical sinusoidal cellular infiltrates along with sheets of atypical mononuclear cells. This finding is in marked contrast to the Antibody (or probe) 1 2 3 4 5 6 BerH2 (CD30) LeuM1 (CD15) LCA (CD45) EMA L26 (CD20) CD3 CD8 MT1 (CD43) UCHL1 (CD45RO) P-cad Leu1 (CD5) Leu2 (CD8) Leu3 (CD4) Leu4 (CD3) bF1 TCRd1 B1 (CD20) B4 (CD19) GrzB PF Leu19 (CD56) LMP1 EBER1 P80 Cb1 111 2 1 1 2 2 2 2 2 11 2 2 11 2 2 2 2 2 1 1 2 2 2 2 2 111 2 2 1 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 11 1 2 1 2 2 2 2 2 11 2 2 11 2 2 2 2 2 1 1 11 2 2 2 2 1 11 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 1 1 2 2 2 2 2 11 11 2 1 2 2 2 2 2 1 2 2 1 2 2 2 2 2 11 1 2 2 2 2 2 1 11 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 LCA: leukocyte common antigen; EMA: epithelial membrane antigen; P-cad: pancadherin; GrzB: granzyme B; PF: perforin; LMP1: latent membrane protein 1; EBER1: EBV-encoded RNA; p80: p80NPM/ALK; Cb1: T cell receptor gene Cb1; 2: positive neoplastic cells , 10%; 1: positive neoplastic cells ^ 10–50%; 11: positive neoplastic cells . 50–80%; 111: positive neoplastic cells . 80%. 976 CANCER February 15, 1999 / Volume 85 / Number 4 TABLE 4 Comparison of Immunohistochemical and In Situ Hybridization Findings among the Six Patients, Anaplastic Large Cell Lymphoma, and Hodgkin Disease Antibody (or probe) The six patients ALCL (n 5 10) Hodgkin’s disease (n 5 19) EMA GrzB PF P-cad LMP1 EBER1 LeuM1(CD15) LCA(CD45) MT1(CD43) UCHL1(CD45RO) P80 4a 4 4 6 0 0 4 1 0 0 0 8 5 8 10 0 0 0 8 6 5 7 1 0 1 10 11 12 14 0 0 0 0 ALCL: anaplastic large cell lymphoma; EMA: epithelial membrane antigen; GrzB: granzyme B; PF: perforin; P-cad: pancadherin; LMP1: latent membrane protein 1; LCA: leukocyte common antigen; p80: p80NPM/ALK. a Number of positive cases. FIGURE 7. Pancadherin staining of an initial lymph node biopsy reveals positive reactivity in many atypical cells (patient 5; 3300). FIGURE 6. Granzyme B staining of an initial lymph node biopsy reveals positive reactivity in most of the atypical cells (patient 4; 3300). biopsies from patients with Hodgkin disease in which sinus infiltration of a few Reed–Sternberg cells was rarely observed. Therefore, we must emphasize the need to examine extensively for sinus involvement in patients with sheets of atypical cells. All of the our patients were positive for BerH2 (CD30). Previous study has indicated that most ALCLs are positive for EMA37 and LCA but are negative for LeuM1. In contrast, most patients with Hodgkin disease are negative for EMA and LCA but are positive for LeuM1. Although immunohistochemical findings of the initial biopsies were mostly consistent with Hodgkin disease phenotypes, subsequent biopsies revealed some change in the phenotypes, namely, EMA positivity in two additional patients. Some patients with ALCL have been reported to be positive for the p80 antibody, which is raised against the NPM/ALK gene.14,38 All of the samples from our patients were negative for this antibody, whereas 7 of 10 samples from patients with classic ALCL were positive. Foss et al.39 reported that almost all T/null ALCLs express perforin40 transcripts and GrzB41– 44 and that Transformation of Hodgkin Disease to ALCL/Mori et al. FIGURE 8. H&E staining of an autopsied lymph node reveals sheets of atypical mononuclear and multinucleated cells (patient 5; 3300). clonal gene rearrangements of the TCR-b chain are observed in 10 of 11 patients with null cell ALCL and in 9 of 10 patients with T-cell ALCL. The current study indicate GrzB expression in four of our six patients and perforin expression in three patients. GrzB and perforin expression was demonstrated in six patients and seven patients with classic ALCL, respectively. Krenacs et al.45 also reported that 76% of patients with ALCL expressed cytotoxic molecules. Oudejans et al.46 found GrzB expression in a portion of the Reed–Sternberg cells found in 11 of 61 patients with Hodgkin disease (18%). However, only one of our patients with Hodgkin disease expressed GrzB or perforin. Therefore, these markers may be useful in differentiating our patients from those with Hodgkin disease, because these molecules are expressed infrequently in Hodgkin disease. The lack of MT1 reactivity9 in our patients sharply contrasts with the frequent reactivity in patients with classic ALCL. Ashton-Key et al.47 reported essentially invariable expression of p-cadherin, a family of cell-to-cell adhesion molecules, in ALCL but less frequent expression 977 in Hodgkin disease. The current study revealed that p-cadherin was expressed in all patients with ALCL and in our patients, but it was absent in 9 of 19 patients with Hodgkin disease. EBER-130,48,49 and/or LMP-150,51 are expressed frequently in Hodgkin disease and are mostly absent in ALCL.52,53 Similarly, EBER-1 positivity was seen in 13 of 19 of our patients with Hodgkin disease. Several previous studies54,55 have indicated that ALCLs may occasionally express EBER-1. However, EBER-1 could not be demonstrated in our set of patients with ALCL or in our current six patients. 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