- Email: [email protected]
Clonal and morphological variation in a posttransplant lymphoproliferative disorder: Evolution from clonal T-cell to clonal B-cell predominance
HUMAN PATHOLOGY
Volume 29, No. 4 (April 1998)
have n o r m a l p53 p r o t e i n e x p r e s s i o n b u t at h a l f the level. It has recently b e e n s h o w n t h a t heterozygous p53 knock-out mice ( + / - ) are m o r e p r o n e to d e v e l o p m e n t a l m a l f o r m a t i o n s t h a n wild-type mice ( + / + ) after x-irradiation in utero, t3 This has b e e n a t t r i b u t e d to a d e c r e a s e d ability o f the + / cells, d a m a g e d b e y o n d repair, to u n d e r g o apoptosis because ind u c e d p53 p r o t e i n levels are d e c r e a s e d c o m p a r e d to + / + mice. O u r results agree well with this idea; that is, only cells with r e d u c e d p53 did n o t die f r o m the R-irradiation w h e r e a s all o t h e r s did, leaving j u s t the clonal progeny. In vitro, e x p e r i e n c e in the e s t a b l i s h m e n t o f cell lines f r o m cervical, a4 thyroid, a5 a n d o t h e r cancers strongly suggests t h a t cells with a b n o r m a l p53 f u n c t i o n have a selective growth advantage, b u t this is the first d e m o n s t r a t i o n o f the clonal e x p a n s i o n in situ o f n o r m a l o r n o n c a n c e r h u m a n cells after d a m a g e to its p53 g e n e (that is, the halving o f the intracellular p53 p r o t e i n level) a n d may illustrate the initial steps in c a r c i n o g e n i c transformation.
Acknowledgment. T h e a u t h o r s are grateful to S h i h o Fujii for technical support. This p u b l i c a t i o n is b a s e d o n r e s e a r c h p e r f o r m e d at the Radiation Effects R e s e a r c h F o u n d a t i o n (RERF), H i r o s h i m a , J a p a n . RERF is a private n o n p r o f i t f o u n d a t i o n f u n d e d equally by t h e J a p a n e s e Ministry o f H e a l t h a n d Welfare a n d t h e U.S. D e p a r t m e n t o f E n e r g y t h r o u g h the National A c a d e m y o f Sciences. REFERENCES 1. Bumpers HL, Natesha RK, Barnwell SP, et al: Multiple and distinct primary cancers:A case report.J Nail Med Assoc86:387-388, 1994
2. Thompson DE, Mabuchi K, Ron E, et al: Cancer incidence in atomic bomb survivors.Part II: Solid tumors, 1958-1987. Radiat Res 137S:17-67, 1994 3. IshikawaY,KatoY, Mori T, et ah e~-particledose to the liverand spleen of Japanese Thorotrast patients. Health Phys 65:497-506, 1993 4. Kato Y, Mori T, Kumatori T: Estimated absorbed dose in tissues and radiation effectsin Japanese Thorotrast patients. Health Plays44:273-279, 1983 (suppl 1) 5. Mori T, Kato Y, Kumatori T, et al: Epidemiologicalfollow-upstudy of Japanese Thorotrast cases--1980.Health Phys44:261-272, 1983 (suppl 1) 6. Kato Y, Ishikawa Y: Portable 22°Rn detector used to assessThorotrast exposure. Health Phys63:119-123, 1992 7. Iwamoto KS, Mizuno T, Ito T, et 21: Feasibility of using decades-old archival tissuesin molecular oncology/epidemiology.AmJ Pathol 149:399-406, 1996 8. Annual of the Pathological Autopsy Cases in Japan: The Japanese Societyof Pathology1974-1989. 9. MclnroyJF, Gonzales ER, MiglioJJ: Measurement of thorium isotopes and 22SRain soft tissuesand bones of a deceased Thorotrast patient. Health Plays 63:54-71, 1992 10. Grillmaier R, Muth H: Radiation dose distribution in lungs of Thorotrast patients. Health Phys20:409-419, 1971 11. Greenblatt MS, Bennett WP, Holstein M, et al: Mutations in the p53 tumor suppressorgene: Clues to cancer etiology.Cancer Res 54:4855-4878, 1994 12. Magewu AN, Jones PA: Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer. Mol Cel Biol 14:4225-4232, 1994 13. Norimura T, Nomoto S, Katsuki M, et al: p53-dependent apoptosis suppressesradiation-induced teratogenesis.Nature Med 2:577-580, 1996 14. Chen TM, Chert CA, Hsieh CY, et al: The state of p53 in primary human cervical carcinoma and its effects in human papillomavirus-immortalized human cervicalcells.Oncogene 8:1511-1518, 1993 15. Wright PA, Lemoine NR, Goretzki PE, et al: Mutation of the p53 gene in a differentiated human thyroid carcinoma cell line, but not in primary thyroid tumors. Oncogene 6:1693-1697, 1991
CLONAL AND MORPHOLOGICAL VARIATION IN A POSI"IrRANSPLANT LYMPHOPROLIFERATIVE DISORDER: EVOLUTION FROM CLONAL T-CELL TO CLONAL B-CELL PREDOMINANCE BEVERLY P. NELSON, MD, JOSEPH LOCKER, MD, PHD, MICHAEL A. NALESNm, MD, JOHN J. FUNG, MD, PHD, AND STEVEN H. SWERDLOW, MD
The majority of posttransplant lymphoproliferative disorders (PTLD) are Epstein-Barr virus (EBV)-associated and of B-cell origin. A much smaller proportion of PTLD are of T-cell origin. We report the clinical, morphological, immunophenotypic, and genotypic results of a unique PTLD, initially diagnosed as immune mediated thrombocytopenia (ITP), which at presentation was predominantly an anaplastic appearing EBV-associated T-cell PTLD and, after reduction in immunosuppression and the administration of antiviral agents, predominantly an EBV-associated plasma cell rich B-cell PTLD. Subsequent chemotherapy resulted in a complete remission. This case has both practical and biological implications. It highlights how PTLD may be misdiagnosed as other entities, how biclonal cases can have different morphological appearances and include both B- and T-cell clones, how PTLD can evolve over time possibly related to immune reconstitution, and why PTLD should be rebiopsied when the disease
From the University" of South Alabama College of Medicine, Mobile, AL and the University of Pittsburgh School of Medicine, Pittsburgh, PA. Address correspondence and reprint requests to Steven H. Swerdlow, MD, Director, Division of Hematopathology, University of Pittsburgh Medical Center Health System, UPMC-Presbyterian, Room C606, 200 Lothrop Street, Pittsburgh, PA 15213-2582. Copyright © 1998 by W.B. Saunders Company 0046-8177/98/2904-002058.00/0
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does not respond to decreased immunosuppression or recurs. HUM PAa~OL 29:416-421. Copyright © 1998 byW.B. Saunders Company Key words: posttransplant lymphoproliferative disorder, biclonal lymphoproliferation, Epstein-Barr virus, immunoglobulin heavy chain, T-cell receptor genes. Abbreviations: PTLD, posttrausplant lymphoproliferatlve disorders; EBV, Epstein-Barr virus; R-S, Reed-Sternberg; WBC, white blood count; RBC, red blood count; Hgb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hgb; MCHC, mean corpuscular hgb concentration; RDW, red cell distribution width; Pit, platelet; ITP, immune mediated thrombocytopeula; CT, computed axial tomography; EBER, Epstein Ban" virus early RNA; JH, immtmoglobin heavy chain gene, joining region; SBA, Southern blot analysis.
P o s t t r a n s p l a n t lymphoproliferative disorders (PTLD) are primarily Epstein-Barr virus (EBV) associated B-cell l y m p h o i d proliferations w h i c h o c c u r after solid o r g a n o r b o n e m a r r o w transplantation. 14 Based largely o n histopathology, the majority can b e categorized as p o l y m o r p h i c or m o n o m o r p h i c PTLD. M Less f r e q u e n t l y cases may r e s e m b l e infectious m o n o nucleosis, a plasmacytoma, m y e l o m a , %cell l y m p h o m a , o r even H o d g k i n ' s disease. 1-14 Plasmacytic hyperplasia is also c o n s i d e r e d a f o r m o f PTLD by some. 3 Classification o f PTLD is p e r f o r m e d in large p a r t b e c a u s e cases o f plasinacytic hyperpla-
CASE STUDIES
sia and polymorphic and polyclonal proliferations are more likely to regress with decreased immunosuppression than the monomorphic monoclonal lesions. Although PTLD with varied histological appearances and with different clones are well described, 1,14,15 the occurrence of both an EBV-associated B-cell PTLD and an EBV-associated T-cell PTLD in a solid organ transplant recipient has, to our knowledge, not been previously reported. We report a patient who had an EBV-associated PTLD with distinct T-cell and B-cell clones. The former clone predominated initially and was associated with the histopathologic appearance of a lymphoma with Reed-Sternberg (R-S)like cells. The latter clone predominated in later specimens after the patient's immunosuppression had been decreased and was associated with a plasma cell rich appearance. Nevertheless, genotypic and i m m u n o p h e n o t y p i c studies showed that the B- and T-cell clones were simultaneously present at most sites. This very unusual case has both practical and biological implications for the diagnosis and understanding of PTLD. It shows that EBV infected B- and T-cell clones can simultaneously be present in a PTLD, how each may be expressed morphologically, and suggests that the clones may respond differently to immune reconstitution after exogenous immunosuppression is decreased. It highlights how PTLD may be misdiagnosed as other benign disorders and shows the importance of performing biopsies of multiple lesions when disease is refractory to therapy or recurs. CASE REPORT This 45-year-old man was diagnosed with ulcerative colitis in 1975 and sclerosing cholangitis in 1988. In February 1990, he developed new onset ascites; cirrhosis was diagnosed and he received a liver transplant in March 1990. His immunosuppressive regimen included Tacrolimus (FK506; Fujisawa, Deerfield, IL) and prednisone. Routine monthly blood work performed in June 1994 showed a platelet count of 8 × 109/L and he was noted to have a lower extremity petechial rash at that time. A complete blood count showed the following: White blood count (WBC), 3.5 × 109/L; red blood cell (RBC), 3.81 × l@2/L; hemoglobin (Hgb) 10.6 g/dL; Hct 32.3; MCV 84.7 fL; MCH 27.9 pg; MCHC 32.9 g/dL; red count distribution width (RDW), 14.0; platelet (PLT), 10 × 109/L. A bone marrow biopsy performed at an outside institution to evaluate the thrombocytopenia showed a normocellular marrow with a mild to moderate megakaryocytic hyperplasia and focal fibrohistiocytic nodules. The results were interpreted as being consistent with an immune mediated thrombocytopenia (ITP) ; the fibrohistiocytic nodules were attributed to a possible drug reaction. He was treated with steroids and platelet transfusions but, because of an inadequate response, was transferred to the University of Pittsburgh Medical Center. At the time of admission, physical examination showed a nonfebrile male with no palpable adenopathy or organomegaly. Laboratory values at admission included WBC, 9.0 × 109/L (differential: neutrophils 80%, monocytes 9%, lymphocytes 7%, atypical lymphocytes 1%, bands 3%); RBC, 3.87 × 1012/L; Hgb, 10.9 g/dL; Hct, 33.0; MCH, 28.1; MCHC, 33.0 g/dL: RDW, 14.5%; MCV, 85.1 fL; Plt, 5 × 109/L. Prothrombin time, activated partial thromboplastin time, and liver function tests were within normal limits. A computed axial tomography (CT) scan performed at admission in June 1994 showed no adenopathy. Vincristine was added to the regimen for treatment of the ITP; however, because of an inadequate response, a splenectomy and liver biopsy were p e r f o r m e d in July 1994.
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The spleen weighed 450 grams and contained a 2.5 cm ill-defined hilar mass as well as multiple 0.1 to 0.6 cm, dark red nodules scattered throughout the parenchyrna. A PTLD was diagnosed. The liver biopsy showed atypical cells in sinusoids, but definite involvement by a PTLD could not be established. A transbronchial lung biopsy performed approximately 1 month after splenectomy to evaluate pulmonary infiltrates showed bronchial mucosal fragments with scattered EBV early RNA (EBER) positive lymphocytes. Based on the diagnosis of PTLD, the patient's Tacrolimus was reduced and he was treated with alpha interferon, ganciclovir, cytogam, and foscarnet. Repeat CT scans performed in August, September, and October 1994 (after antiviral therapy and a decrease in immunosuppression) showed persistent retroperitoneal and mesenteric adenopathy. Approximately 3 months after splenectomy (October 1994), repeat liver function tests showed alkaline phosphatase of 496 I U / L (nL 40-125) and total bilirubin 8.7 m g / d L (nL 0.2 to 1.3). The aspartate aminotransferase and alanine aminotransferase were within normal limits. A bone marrow biopsy from September 1994 and liver biopsies performed in September and October 1994 showed involvement by PTLD. Because of continued abnormal linear function tests (LFT), histological evidence of persistent liver involvement by PTLD, and radiologic evidence of PTLD progression, he received 6 cycles of CHOP (cyclophosphamide, daunorubicin, vincristine, prednisone) chemotherapy between October 1994 and March 1995. CT scans performed in April and July 1995 showed no evidence of adenopathy. The FK506 was restarted in August 1995. The patient continues to have an adequately functioning allograft liver with no clinical or radiological evidence of PTLD 34 months after initial diagnosis. METHODS
Histology Sections of spleen, bone marrow, liver, and transbronchial lung biopsy specimens fixed in formalin or B5 fixative, routinely processed, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff stains were reviewed.
Immunohistochemistry Immunoperoxidase staining was performed on 4 pm deparaffinized tissue sections using the Ventana automated immunostainer (Tucson, AZ) and their DAB detection kit. The primary antibodies were directed against the epithelial membrane antigen, CD20 (L26), CD30 (BetH2), CD3 (polyclonal) (Dako, Carpinteria, CA), and K and )t light chains (Neomarkers, Fremont, CA).
RNA In Situ Hybridization In situ hybridization for K and X light chain mRNA and EBER-1 RNA was performed using a modification of the method of Weiss et a116,17 as described by Aguilera et aP s and biotinylated oligonucleotide probes for K (4 oligonucleotides) and X (6 oligonucleotides) mRNA and the EBER-1 probe for EBV-RNA (30 base digoxigenin-labeled oligonucleotide; Research Genetics, Huntsville, AL). Gcnotypic Studies Bone marrow aspirate suspended in ethylinediaminetetraacetic acid and fresh tissue from the spleen and liver biopsy were obtained. Methods used for Southern blot analysis (SBA) and DNA probes for the immunoglobulin hea W chain gene
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Volume 29, No. 4 (April 1998)
j o i n i n g region (JH) and EBV terminal repeat region have been described previously.19 The T-cell receptor [3-chain probe was described by Weiss et al. 2° RESULTS HistopathologT/Immunophenotype Retrospective review of the initial bone marrow biopsy, interpreted elsewhere as ITP, showed scattered lymphoid aggregates with variable numbers of admixed histiocytes and some with rare R-S-like cells (Table 1). Most lymphocytes were CD3 and CD8 positive. The R-S-like cells were CD15, CD30, and EBV-positive. Histological sections from the spleen showed an extensive red pulp infiltrate and small white pulp nodules composed of small lymphocytes, plasma cells, histiocytes (some showing erythrophagocytosis), and conspicuous large mononuclear and multinucleated cells with prominent nucleoli resembling R-S cells and R-S variants (Fig 1A). Small foci of more numerous plasma ceils were also present. Immunohistochemical stains supported a T-cell origin for the large cells (Fig 1B, Table 1). In situ hybridization studies showed small clusters of K light chain class restricted plasma cells (Fig 1C). The vast majority of R-S-like cells, occasional plasma cells, and variably sized lymphoid ceils were EBV-positive. The liver biopsy performed at the time of splenectomy showed scattered atypical lymphoid cells within the sinusoids but was not considered diagnostic of a PTLD. A subsequent liver biopsy, 3 months later, showed portal tracts expanded by a lymphoplasmacytic infiltrate with numerous EBV-positive plasma cells that showed a marked K predominance. The repeat bone marrow biopsy from September 1994 showed focal relatively homogeneous aggregates of plasma cells throughout the medullary cavity (Fig 2). Some plasma cell aggregates had many admixed lymphocytes, and some were associated with granulomas. The plasma cells showed a marked K predominance. Moderate numbers of small CD3positive lymphocytes, mostly associated with the plasma cell
TABLE 1. H i s t o p a t h o l o g y a n d I m m u n o p h e n o t y p i c Results BM* (6/94) Histopathology R-S-like cells Plasma cells Antigen Total/R-S like cells§ Kappa Lambda LCA CD20 (L26) CD79a CD3 CD8 CD15(LeuM1) CD30(BerH2) EMA EBER-ISH
Spleen (7/94)
Liver (9-10/94)
BM (9/94)
+* +
+ + +t + +j-
+ +++
+++
+/+/+++/+ -/ND +++/+++/+++/+ -/++ -+/-4+/++
++/+/+++/-+ +/+/+++/+++ +++/+/+ +++/+++ +/+ +/+++
+++/+/ND +/ND ++/ND -/++/+ ND ++
+++/+/ND +/ND ++/+ ++/+++/ND ND +++
FIGURE 1. T-cell predominant PTLD with Hodgkin-like features in spleen. (A) Note the pleomorphic diffuse infiltrate including large binucleated and multinucleated cells resembling R-S cells, (B) These ceils stained with the polyclonaI CD3 antibody. (C) In situ hybridization stains showed small foci of K positive plasma cells (left) in areas in which the X stain was negative (right). ([A] Hematoxylin and eosin [H&E] stain. [B] CD3 immunoperoxidase stain with hematoxylin counterstain, [C) in situ hybridization stain for K and X with hematoxylin counterstain).
Abbreviations: R-S, Reed-Sternberg; LCA, leukocyte common antigen; EMA, epithelial membrane antigen; EBER, Epstein Barr virus encoded RNA;ISH, in situ hybridization. *+ = scattered (up to a few seen on scanning). t + + = moderate (easilyfound). ~.+ + + = numerous (present in every field). §Results shown separately for total lymphocyte population and R-S-like ceils.
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aggregates, were seen. Rare R-S--like cells were also CD3positive. Both lymphocytes and plasma cells showed positivity for EBV. Genotypic Studies The genotypic studies, as summarized in Table 2, showed clonal TCR-[3 chain gene rearrangement in the spleen and
CASE STUDIES
FIGURE 2. Plasma cell rich PTLD involving the bone marrow (September 1994). Note the numerous plasma cells replacing portions of the medullary space. These cells showed monetypic ~ light chain expression. There are some admixed histiocytes, (H&E stain.) subsequent bone marrow biopsy from September 1994 (Fig 3). The immunoglobulin heavy chain gene was clonally rearranged in the bone marrow whereas the spleen and liver showed only germline bands. EBV terminal repeat analysis showed three clonal bands in the spleen and two or three clonal bands in the liver and bone marrow. DISCUSSION The case reported here broadens the concept of how PTLD can show a histological, phenotypic, and genotypic spectrum of disease within an individual patient and how PTLD can evolve over time. The patient had an EBV associated T-cell PTLD at some sites and a plasma cell rich EBV-associated B-cell PTLD that was most prominent at other sites. Ancillary studies however supported the coexistence of both PTLD at most sites. The T-cell PTLD with some Hodgkin'slike features was most apparent in the initial bone marrow biopsy and in the spleen. The increasing predominance of the B-cell clonal proliferation seen in the subsequent marrow and liver biopsy specimens occurred after the reduction in immunosuppression and the administration of antiviral agents. The initial T-cell PTLD, which bore some resemblance to Hodgkin's disease, seems similar to some of the other T-cell PTLDs that have been reported. Fourteen percent of all PTLD have been reported to be of T-cell origin.8 When tested, most have shown clonal T-cell receptor gene rearrangement (8 of 9 cases s) similar to the current case. EBV positivity, as seen here, has been reported in at least seven other T-cell PTLD. 8,12,2I The reported cases of T-cell PTLD show a histopathologic spectrum including pleomorphic and anaplastic large cell lymphomas, one of which had CD30 positivity and splenic
TABLE 2. Genotype
BM (6/94)
TCR-[3 JH EBV (terminal repeat analysis)
ND ND ND
involvement.8 The latter case was not clearly associated with EBV. The plasma cell rich B-cell P ~ D principally involved the allograft liver and bone marrow specimens obtained after the patient's immunosuppression had been reduced. These lesions show architectural destruction and are predominantly composed of plasma cells and plasmacytoid cells with few transformed cells, is It is of interest that although the B-cell clone and numerous plasma cells predominated only in the later PTLD, a small K restricted plasma cell component was present in the splenectomy specimen with the T-cell PTLD. Although not seen with the SBA using the JH probe, the EBV terminal repeat analysis supports the observation that the major clone present in the later marrow and liver biopsy specimens is present as a minor clone in the spleen. Conversely, the later marrow which histologically and phenotypically has a predominant B-cell clone, also shows evidence of an identical T-cell clone. It is uncertain why the major EBV band present in the spleen is hardly detected in the later marrow because the latter includes a component of the original T-cell PTLD. These morphological, immunophenotypic, and genotypic results support the presence of a multiclonal PTLD in which the more anaplastic T-cell component showed an apparent significant response to decreasing the patient's immunosuppression; however, the more indolent appearing clonal plasma cell rich component showed an apparent growth advantage. The patient ultimately required conventional chemotherapy for control of his PTLD. The current case shares some features with that of a renal transplant recipient who developed a cutaneous T-cell lymphoma followed by a nodal lymphoma containing both B- and T-cell clones and EBV.22 The phenotype a n d / o r genotype of the EBV-positive cells was not clear, and EBV was not shown in the cutaneous T-cell PTLD. A second renal transplant recipient developed a scalp-based EBV-positive B-cell PTLD (T-cell clonality not investigated) and a subsequent T-cell clonal population was shown in the peripheral blood. 12It is unknown if EBVwas present in the T-cell clone. A third renal transplant recipient showed allograft involvement by a phenotypically B-cell PTLD with genotypic evidence of both immunoglobulin and T-cell receptor gene rearrangement. 21 This patient's PTLD, however, did not show two histologically distinct lesions and there is no evidence that the gene rearrangements were in two distinct clones. Although seemingly unique, the existence of a PTLD such as reported here is not surprising given the many previous observations that individual patients may have distinct B-cell clones at multiple or even single sites. 1,I5,19 Furthermore, PTLDs are known to relapse sometimes with morphologically a n d / o r clonally distinct disease. 5 The current case, however, highlights the need to consider the presence of simultaneous or subsequent B- and T-cell clones and the need to biopsy different sites of disease particularly when patients
G e n o t y p i c Studies
Spleen (7/94)
Liver (9-10/94)
BM (9/94)
Clonal & polyclonal pattern* ND Clonal & polyclonal pattern* Germline Germline Clonal (faint) 1 major & 2 minor clonal 1 major & 1 minor clonal band]- 1 major & 2 minor clonal bands]bandst
Abbreviations: TCR-13,T-cellreceptor beta chain gene;JH, immunoglobinheavy chain gene,joining region; EBV,Epstein Barr virus. *The TCR-[3clone in the spleen and the bone marrow show similar electrophoretic mobility suggesting that they represent the same clone at both sites. ]-The two minor EBV clones in the spleen are in similar positions as the major and minor clones in the liver and bone marrow.
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HUMAN PATHOLOGY
Volume 29, No. 4 (April 1998)
1 2 3
EcoRV
Jn BglII
EBV-Terminus BamHI
FIGURE 3. Genotypic results using SBA. The arrows at the far left indicate the bands representing common clonal rearrangement of the TCR-I~ chain gene in the spleen and bone marrow (September 1994). Immunoglobulin heavy chain rearrangement was identified in the marrow (September 1994), but not the spleen (arrow in middle panel). EBV terminal repeat analysis showed major (#1 in panel on right) and 2 minor clonal bands (#2 and #3) in the spleen. The liver and bone marrow e a c h show a major and a minor clonal band although a faint band in position 1 may also be present in marrow. Note the similar location of one minor EBV clonal band at all three sites (#3). The major EBV clonai bands of the liver and bone marrow are also in a similar position to a minor clonal band in the spleen (#2).
do not respond to decreased immunosuppression or when disease recurs. Finally, this patient was initially diagnosed as having idiopathic thrombocytopenic purpura even after a bone marrow biopsy. Thrombocytopenic purpura with increased marrow megakaryocytes can follow EBV infectionZS; however, this case proved to be a PTLD. It therefore emphasizes the need to always maintain a high degree of suspicion for PTLD in patients after transplantation. REFERENCES 1. Nalesnik MA, Jaffe R, Starzl TE, et ah The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine Aprednisone immunosuppression. AmJ Pathol 133:173-92, 1988 2. Frizzera G, Hanto DW, Gajl-Peczalska KJ, et al: Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients. Cancer Res 41:4262-4279, 1981
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3. Kxlowles DM, Cesarman E, Chadburn A, et al: Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood 85:552-565, 1995 4. Swerdlow SH: Classification of the posttransplant lymphoproliferative disorders: From the past to the present. Semin Diagn Pathol 14:2-7, 1997 5. Wu T-T, Swerdlow 8H, Locker J, et al: Recurrent Epstein-Barr-Virus associated lesions in organ transplant recipients. HUM PATHOL27:157-64, 1996 6. Waller EK, Ziemianska M, Bangs CD, et al: Characterization of posttransplant lymphomas that express T-cell-associated markers: Immunophenotypes, molecular genetics, cytogenetics, and heterotransplantation in severe combined immunodeficient mice. Blood 82:247-61, 1993 7. Ross CW, Schnitzer B, Sheldon S, et al: Gamma/Delta T-cen posttransplantation lymphoproliferative disorder primarily in the spleen. Am J Clin Pathol 102:310-315, 1994 8. vanGorp J, Doornewaard H, Verdonck LF, et al: Posttransplant T-cell lymphoma. Report of three cases and a review of the literature. Caiacer 73:3064-3072, 1994 9. Kaplan MA, JacobsonJO, FerryJA, et al: T-cell lymphoma of the vulva in
CASE STUDIES a renal allograft recipient with associated henmphagocytosis. AmJ Snrg Pathol 17:842-849, 1993 10. Morrison VA, Dunn DL, ManivelJC, et al: Clinical characteristics of post-transplant lymphoproliferative disorders. AmJ Med 97:14-24, 1994 11. Nalesnik MA, Randhawa P, Demetris AJ, et al: Lymphoma resembling Hodgkin disease after posttransplant lymphoproliferative disorder in a liver transplant recipient. Cancer 72:2568-2573, 1993 12. Frankel AH, Tbompson M, Vulliamy T, et al: A T cell clone in association with an Epstein-Barr virus-related B cell lymphoma (Letters to Editor). Transplantation 52:1108-1110, 1991 13. SwerdlowSH: Posttransplant lymphoproliferative disorders: A working classification. Curt Diagn Pathol 4:29-36, 1997 14. SwerdlowSH:Post-transplant lymphoproliferative disorders:Amorpholo,~c, phenotypic and genotypic spectrum of disease. Histopathology 20:373385, 1992 15. Chadburn A, Cesarman E, YiFang L, et al: Molecular genetic analysis demonstrates that multiple posttransplant lymphoproliferative disorders occurring in one anatomic site in a single patient represent distinct primary lymphoid neoplasms. Cancer 75:2747-2756, 1995 16. WeissLM, Movahed LA, Chen Y-Y,et al: Detection ofimmunoglobulin light-chain mRNA in lymphoid tissues using a practical in situ hybridization method. AmJ Patho1137:979-988, 1990
17, Weiss LM, Chen Y-Y,Liu X-F, et al: Epstein-Barr Virus and Hodgkin's Disease. A correlative in sire hybridization and polymerase chain reaction study. AmJ Pathol 139:1259-1265,1991 18. Aguilera NS, Kapadia SB, Nalesnik NLA,et ah Extramednllary plasmacytoma of the head and neck: Use of paraffin sections to assessclonalitywith in situ hybridization, growth fraction, and the presence of Epstein-Barr virus. Mod Pathol 8:503-508, 1995 19. Locker J, Nalesnik M: Molecular genetic analysis of lymphoid tumors arising after organ transplantation. AmJ Pathol 135:977-987, 1989 20. WeissLM, Hu E, Wood GS, et al: Clonal rearrangements of the T-cell receptor gene in mycosis fangoides and dermatopathic lymphadenopathy. N EnglJ Med 313:539-544, 1985 21. Hollingsworth HC, Stetler-StevensonM, Gagneten D, et al: Immunodeficiency-associated malignant lymphoma. Three cases showing genotypic evidence of both 2"-and B-celllineages. AmJ Surg Pathol 18:1092-1101, I994 22. Euvrard S, Noble CP, Nanitakis J, et ah Brief report: Successive occurrence of T-cell and B-cell lymphomas after renal transplantation in a patient with multiple cutaneous squamous-cen carcinomas. N Engl J Med 327:1924-1926, 1992 23. MatsukawaY, Okano M, Ishikawa N, et aI: Severe thrombocytopenic purpura associated with primary Epstein-Barr virus infection. J Infect 29:107109, 1994 (letter)
ENHANCED EXPRESSION OF INTERLEUKIN-6 IN BONE AND SERUM OF METASTATIC RENAL CELL CARCINOMA BERNARD PAULE, MD, DIDIER CLERC, MD, CHRISTIANE RUDANT, COLETTE COULOMBEL, LAURENCE BONHOMME-FAWRE, MD, JEANINE QUILIARD, MD, AND MICHEL BISSON, MD
Interleukin-6 (IL-6) is produced by renal cell carcinoma (RCC) cell lines and primary tumors. Using immunohlstochemlcal staining in two RCC patients with hypercaleemia and high serum levels of free and total II~6, we showed expression of IL-6 in metastatic bone tissue. The role of IL-6 ha hypercalcemia and bone resorption would suggest that bisphosphonates or dexamethasone could be useful as adjuvant therapy for IL-6 dependant bone metastases which fail to respond to interferon alpha (IFN) ol 2a and all traxts retinoic add
(ATRA). HUM PATHOL29:421424. Copyright © 1998 byW.B. Saunders Company Key words: Interleuldn-6, bone metastasis, renal cell carcinoma, immunohistochemistry. Abbreviations: IL-6, Interleukin 6; RCC, renal cell carcinoma; IFN, interferon; ATRA, All trans retinoic acid; sIL-6R, Soluble receptor to IL-6; PBS, Phosphate-buffered saline; PTHrP, Parathyroid hormone related peptide.
I n t e r l e u k i n - 6 (IL-6) is a p l e i o t r o p i c c y t o k i n e s y n t h e t i z e d b y b o t h n o r m a l a n d t u m o r a l cells. 1 I t is p r o d u c e d b y r e n a l cell c a r c i n o m a ( R C C ) c e l l l i n e s 2,3 a n d i n patients with primary RCC, 4 and has been shown to act as a n R C C a u t o c r i n e g r o w t h f a c t o r . 5,6 E l e v a t e d s e r u m l e v e l s o f IL-6 b e f o r e t r e a t m e n t a r e c o r r e l a t e d w i t h shorter survival and lack of response to immunotherapy, v In two patients with advanced stage RCC and h y p e r c a l c e m i a , w e e v a l u a t e d IL-6 e x p r e s s i o n i n b o n e m e t a s t a t i c t i s s u e s a n d s e r u m l e v e l s o f f r e e IL-6 o r IL-6 b o u n d t o its s o l u b l e r e c e p t o r ( s I L - 6 R ) .
L a s e g u e ' s sign, clinical e x a m i n a t i o n was u n r e m a r k a b l e . H e h a d raised e r y t h r o c y t e s e d i m e n t a t i o n rate (ESR) (90 m m 1st h o u r ) , C-reactive p r o t e i n (CRP: 149 m g / L , N L < 112 m g / L ) a n d calcemia (2,90 m m o l / L , NL: 2,25-2,60 m m o l / L ) ; comp l e t e b l o o d count, c r e a t i n e m i a , a n d liver enzymes were n o r m a l . S e r u m i n t a c t p a r a t h y r o i d h o r m o n e (10 p g / m L , NL: 10 to 65 p g / m L ) , 25 h y d r o x y v i t a m i n D (7,2 n g / L , NL: 8 to 30 n g / m L ) were lowered a n d calcitriol (27 p g / m L , NL: 20 to 80 p g / m L ) was n o r m a l . R a d i o g r a p h s disclosed a n o r m a l l u m b a r s p i n e a n d a r i g h t sacral osteolysis. Sacrum, r i g h t t r o c h a n t e r i c , a n d ilio-ischiatic h o t spots were revealed o n t e c h n e t i u m 9 9 m (99mWc) skeletal scintigraphy. Lumbo-sacral, a b d o m i n a l a n d t h o r a c i c c o m p u t e r i z e d t o m o g r a p h i c studies respectively docum e n t e d (1) a large (6,5 c m × 7 c m × 4,5 cm) lyric L5 a n d $1 v e r t e b r a l a n d sacral lesion i n v a d i n g t h e spinal canal, compressi n g t h e t h e c a l sac a n d a d h e r i n g to t h e iliac vessels; (2) a r e n a l tissue mass w h i c h was e n h a n c e d after injection; (3) five r i g h t p u l m o n a r y i n f r a c e n t i m e t r i c n o d u l e s a n d a 6 5 m m large subp l e u r a l mass in t h e p o s t e r o b a s a l s e g m e n t o f t h e left lower lobe. A sacral biopsy was p e r f o r m e d . P a t h o l o g i c a l study c o n c l u d e d to a p o o r l y d i f f e r e n t i a t e d a d e n o c a r c i n o m a with clear cells in favor o f a r e n a l origin. H y p e r c a l c e m i a a n d p a i n resolved w h e n t r e a t e d by p a m i d r o n a t e infusions, oral corticosteroids, a n d m o r p h i n e . S u b c u t a n e o u s i n t e r f e r o n (IFN) c~2a (Roferon-A, H o f f m a n n - R o c h e , France, 18 million units 3 days a week) a n d oral all trans r e t i n o i c acid (ATRA, H o f f m a n n Roche, France, 100 m g / m 2 / d a y given in t h r e e equally divided doses) were i n s t a u r e d o n D e c e m b e r 13th as described, s O n e m o n t h later, r e c r u d e s c e n t p a i n c a u s e d by sacral t u m o r progression led to e m b o l i z a t i o n t h e n L5 v e r t e b r a l a n d s a c r u m r a d i o t h e r a p y (45 Gy, five f r a c t i o n s / w e e k , 2 G y / f r a c t i o n ) .
PATIENTS AND M E T H O D S :
Case 1 A 55-year-old m a n was a d m i t t e d in D e c e m b e r 1993 with a 2 - m o n t h history o f r i g h t S1 sciatica a n d r e c e n t loss in weight o f 8 kilograms. A p a r t f r o m a l u m b a r stiffness a n d a r i g h t From the Service de Rhumatologie, H6pital de Bic~tre, Le Kremlin-Bic6tre, France; the UFR Biom6dicale UPR 37, R6gulation de l'expression des g~nes, Paris, France; the Service de Pharmacie, D6partement de Pharmacologie, H6pital Paul Brousse, Villejuif, France; and the Service d'Anatomo-pathologie, H6pital de Bic&re, Le Kremlin-BicStre, France. Address correspondence and reprint requests to Bernard Paule, MD, Service de Rhumatologie, H6pital de BicStre, 78 rue dn G6n~ral Leclerc, 94 275 Le Kremlin Bic6tre, France. Copyright © 1998 by W.B. Sannders Company 0046-8177/98/2904-000X$8.00/0
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have n o r m a l p53 p r o t e i n e x p r e s s i o n b u t at h a l f the level. It has recently b e e n s h o w n t h a t heterozygous p53 knock-out mice ( + / - ) are m o r e p r o n e to d e v e l o p m e n t a l m a l f o r m a t i o n s t h a n wild-type mice ( + / + ) after x-irradiation in utero, t3 This has b e e n a t t r i b u t e d to a d e c r e a s e d ability o f the + / cells, d a m a g e d b e y o n d repair, to u n d e r g o apoptosis because ind u c e d p53 p r o t e i n levels are d e c r e a s e d c o m p a r e d to + / + mice. O u r results agree well with this idea; that is, only cells with r e d u c e d p53 did n o t die f r o m the R-irradiation w h e r e a s all o t h e r s did, leaving j u s t the clonal progeny. In vitro, e x p e r i e n c e in the e s t a b l i s h m e n t o f cell lines f r o m cervical, a4 thyroid, a5 a n d o t h e r cancers strongly suggests t h a t cells with a b n o r m a l p53 f u n c t i o n have a selective growth advantage, b u t this is the first d e m o n s t r a t i o n o f the clonal e x p a n s i o n in situ o f n o r m a l o r n o n c a n c e r h u m a n cells after d a m a g e to its p53 g e n e (that is, the halving o f the intracellular p53 p r o t e i n level) a n d may illustrate the initial steps in c a r c i n o g e n i c transformation.
Acknowledgment. T h e a u t h o r s are grateful to S h i h o Fujii for technical support. This p u b l i c a t i o n is b a s e d o n r e s e a r c h p e r f o r m e d at the Radiation Effects R e s e a r c h F o u n d a t i o n (RERF), H i r o s h i m a , J a p a n . RERF is a private n o n p r o f i t f o u n d a t i o n f u n d e d equally by t h e J a p a n e s e Ministry o f H e a l t h a n d Welfare a n d t h e U.S. D e p a r t m e n t o f E n e r g y t h r o u g h the National A c a d e m y o f Sciences. REFERENCES 1. Bumpers HL, Natesha RK, Barnwell SP, et al: Multiple and distinct primary cancers:A case report.J Nail Med Assoc86:387-388, 1994
2. Thompson DE, Mabuchi K, Ron E, et al: Cancer incidence in atomic bomb survivors.Part II: Solid tumors, 1958-1987. Radiat Res 137S:17-67, 1994 3. IshikawaY,KatoY, Mori T, et ah e~-particledose to the liverand spleen of Japanese Thorotrast patients. Health Phys 65:497-506, 1993 4. Kato Y, Mori T, Kumatori T: Estimated absorbed dose in tissues and radiation effectsin Japanese Thorotrast patients. Health Plays44:273-279, 1983 (suppl 1) 5. Mori T, Kato Y, Kumatori T, et al: Epidemiologicalfollow-upstudy of Japanese Thorotrast cases--1980.Health Phys44:261-272, 1983 (suppl 1) 6. Kato Y, Ishikawa Y: Portable 22°Rn detector used to assessThorotrast exposure. Health Phys63:119-123, 1992 7. Iwamoto KS, Mizuno T, Ito T, et 21: Feasibility of using decades-old archival tissuesin molecular oncology/epidemiology.AmJ Pathol 149:399-406, 1996 8. Annual of the Pathological Autopsy Cases in Japan: The Japanese Societyof Pathology1974-1989. 9. MclnroyJF, Gonzales ER, MiglioJJ: Measurement of thorium isotopes and 22SRain soft tissuesand bones of a deceased Thorotrast patient. Health Plays 63:54-71, 1992 10. Grillmaier R, Muth H: Radiation dose distribution in lungs of Thorotrast patients. Health Phys20:409-419, 1971 11. Greenblatt MS, Bennett WP, Holstein M, et al: Mutations in the p53 tumor suppressorgene: Clues to cancer etiology.Cancer Res 54:4855-4878, 1994 12. Magewu AN, Jones PA: Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer. Mol Cel Biol 14:4225-4232, 1994 13. Norimura T, Nomoto S, Katsuki M, et al: p53-dependent apoptosis suppressesradiation-induced teratogenesis.Nature Med 2:577-580, 1996 14. Chen TM, Chert CA, Hsieh CY, et al: The state of p53 in primary human cervical carcinoma and its effects in human papillomavirus-immortalized human cervicalcells.Oncogene 8:1511-1518, 1993 15. Wright PA, Lemoine NR, Goretzki PE, et al: Mutation of the p53 gene in a differentiated human thyroid carcinoma cell line, but not in primary thyroid tumors. Oncogene 6:1693-1697, 1991
CLONAL AND MORPHOLOGICAL VARIATION IN A POSI"IrRANSPLANT LYMPHOPROLIFERATIVE DISORDER: EVOLUTION FROM CLONAL T-CELL TO CLONAL B-CELL PREDOMINANCE BEVERLY P. NELSON, MD, JOSEPH LOCKER, MD, PHD, MICHAEL A. NALESNm, MD, JOHN J. FUNG, MD, PHD, AND STEVEN H. SWERDLOW, MD
The majority of posttransplant lymphoproliferative disorders (PTLD) are Epstein-Barr virus (EBV)-associated and of B-cell origin. A much smaller proportion of PTLD are of T-cell origin. We report the clinical, morphological, immunophenotypic, and genotypic results of a unique PTLD, initially diagnosed as immune mediated thrombocytopenia (ITP), which at presentation was predominantly an anaplastic appearing EBV-associated T-cell PTLD and, after reduction in immunosuppression and the administration of antiviral agents, predominantly an EBV-associated plasma cell rich B-cell PTLD. Subsequent chemotherapy resulted in a complete remission. This case has both practical and biological implications. It highlights how PTLD may be misdiagnosed as other entities, how biclonal cases can have different morphological appearances and include both B- and T-cell clones, how PTLD can evolve over time possibly related to immune reconstitution, and why PTLD should be rebiopsied when the disease
From the University" of South Alabama College of Medicine, Mobile, AL and the University of Pittsburgh School of Medicine, Pittsburgh, PA. Address correspondence and reprint requests to Steven H. Swerdlow, MD, Director, Division of Hematopathology, University of Pittsburgh Medical Center Health System, UPMC-Presbyterian, Room C606, 200 Lothrop Street, Pittsburgh, PA 15213-2582. Copyright © 1998 by W.B. Saunders Company 0046-8177/98/2904-002058.00/0
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does not respond to decreased immunosuppression or recurs. HUM PAa~OL 29:416-421. Copyright © 1998 byW.B. Saunders Company Key words: posttransplant lymphoproliferative disorder, biclonal lymphoproliferation, Epstein-Barr virus, immunoglobulin heavy chain, T-cell receptor genes. Abbreviations: PTLD, posttrausplant lymphoproliferatlve disorders; EBV, Epstein-Barr virus; R-S, Reed-Sternberg; WBC, white blood count; RBC, red blood count; Hgb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hgb; MCHC, mean corpuscular hgb concentration; RDW, red cell distribution width; Pit, platelet; ITP, immune mediated thrombocytopeula; CT, computed axial tomography; EBER, Epstein Ban" virus early RNA; JH, immtmoglobin heavy chain gene, joining region; SBA, Southern blot analysis.
P o s t t r a n s p l a n t lymphoproliferative disorders (PTLD) are primarily Epstein-Barr virus (EBV) associated B-cell l y m p h o i d proliferations w h i c h o c c u r after solid o r g a n o r b o n e m a r r o w transplantation. 14 Based largely o n histopathology, the majority can b e categorized as p o l y m o r p h i c or m o n o m o r p h i c PTLD. M Less f r e q u e n t l y cases may r e s e m b l e infectious m o n o nucleosis, a plasmacytoma, m y e l o m a , %cell l y m p h o m a , o r even H o d g k i n ' s disease. 1-14 Plasmacytic hyperplasia is also c o n s i d e r e d a f o r m o f PTLD by some. 3 Classification o f PTLD is p e r f o r m e d in large p a r t b e c a u s e cases o f plasinacytic hyperpla-
CASE STUDIES
sia and polymorphic and polyclonal proliferations are more likely to regress with decreased immunosuppression than the monomorphic monoclonal lesions. Although PTLD with varied histological appearances and with different clones are well described, 1,14,15 the occurrence of both an EBV-associated B-cell PTLD and an EBV-associated T-cell PTLD in a solid organ transplant recipient has, to our knowledge, not been previously reported. We report a patient who had an EBV-associated PTLD with distinct T-cell and B-cell clones. The former clone predominated initially and was associated with the histopathologic appearance of a lymphoma with Reed-Sternberg (R-S)like cells. The latter clone predominated in later specimens after the patient's immunosuppression had been decreased and was associated with a plasma cell rich appearance. Nevertheless, genotypic and i m m u n o p h e n o t y p i c studies showed that the B- and T-cell clones were simultaneously present at most sites. This very unusual case has both practical and biological implications for the diagnosis and understanding of PTLD. It shows that EBV infected B- and T-cell clones can simultaneously be present in a PTLD, how each may be expressed morphologically, and suggests that the clones may respond differently to immune reconstitution after exogenous immunosuppression is decreased. It highlights how PTLD may be misdiagnosed as other benign disorders and shows the importance of performing biopsies of multiple lesions when disease is refractory to therapy or recurs. CASE REPORT This 45-year-old man was diagnosed with ulcerative colitis in 1975 and sclerosing cholangitis in 1988. In February 1990, he developed new onset ascites; cirrhosis was diagnosed and he received a liver transplant in March 1990. His immunosuppressive regimen included Tacrolimus (FK506; Fujisawa, Deerfield, IL) and prednisone. Routine monthly blood work performed in June 1994 showed a platelet count of 8 × 109/L and he was noted to have a lower extremity petechial rash at that time. A complete blood count showed the following: White blood count (WBC), 3.5 × 109/L; red blood cell (RBC), 3.81 × l@2/L; hemoglobin (Hgb) 10.6 g/dL; Hct 32.3; MCV 84.7 fL; MCH 27.9 pg; MCHC 32.9 g/dL; red count distribution width (RDW), 14.0; platelet (PLT), 10 × 109/L. A bone marrow biopsy performed at an outside institution to evaluate the thrombocytopenia showed a normocellular marrow with a mild to moderate megakaryocytic hyperplasia and focal fibrohistiocytic nodules. The results were interpreted as being consistent with an immune mediated thrombocytopenia (ITP) ; the fibrohistiocytic nodules were attributed to a possible drug reaction. He was treated with steroids and platelet transfusions but, because of an inadequate response, was transferred to the University of Pittsburgh Medical Center. At the time of admission, physical examination showed a nonfebrile male with no palpable adenopathy or organomegaly. Laboratory values at admission included WBC, 9.0 × 109/L (differential: neutrophils 80%, monocytes 9%, lymphocytes 7%, atypical lymphocytes 1%, bands 3%); RBC, 3.87 × 1012/L; Hgb, 10.9 g/dL; Hct, 33.0; MCH, 28.1; MCHC, 33.0 g/dL: RDW, 14.5%; MCV, 85.1 fL; Plt, 5 × 109/L. Prothrombin time, activated partial thromboplastin time, and liver function tests were within normal limits. A computed axial tomography (CT) scan performed at admission in June 1994 showed no adenopathy. Vincristine was added to the regimen for treatment of the ITP; however, because of an inadequate response, a splenectomy and liver biopsy were p e r f o r m e d in July 1994.
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The spleen weighed 450 grams and contained a 2.5 cm ill-defined hilar mass as well as multiple 0.1 to 0.6 cm, dark red nodules scattered throughout the parenchyrna. A PTLD was diagnosed. The liver biopsy showed atypical cells in sinusoids, but definite involvement by a PTLD could not be established. A transbronchial lung biopsy performed approximately 1 month after splenectomy to evaluate pulmonary infiltrates showed bronchial mucosal fragments with scattered EBV early RNA (EBER) positive lymphocytes. Based on the diagnosis of PTLD, the patient's Tacrolimus was reduced and he was treated with alpha interferon, ganciclovir, cytogam, and foscarnet. Repeat CT scans performed in August, September, and October 1994 (after antiviral therapy and a decrease in immunosuppression) showed persistent retroperitoneal and mesenteric adenopathy. Approximately 3 months after splenectomy (October 1994), repeat liver function tests showed alkaline phosphatase of 496 I U / L (nL 40-125) and total bilirubin 8.7 m g / d L (nL 0.2 to 1.3). The aspartate aminotransferase and alanine aminotransferase were within normal limits. A bone marrow biopsy from September 1994 and liver biopsies performed in September and October 1994 showed involvement by PTLD. Because of continued abnormal linear function tests (LFT), histological evidence of persistent liver involvement by PTLD, and radiologic evidence of PTLD progression, he received 6 cycles of CHOP (cyclophosphamide, daunorubicin, vincristine, prednisone) chemotherapy between October 1994 and March 1995. CT scans performed in April and July 1995 showed no evidence of adenopathy. The FK506 was restarted in August 1995. The patient continues to have an adequately functioning allograft liver with no clinical or radiological evidence of PTLD 34 months after initial diagnosis. METHODS
Histology Sections of spleen, bone marrow, liver, and transbronchial lung biopsy specimens fixed in formalin or B5 fixative, routinely processed, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff stains were reviewed.
Immunohistochemistry Immunoperoxidase staining was performed on 4 pm deparaffinized tissue sections using the Ventana automated immunostainer (Tucson, AZ) and their DAB detection kit. The primary antibodies were directed against the epithelial membrane antigen, CD20 (L26), CD30 (BetH2), CD3 (polyclonal) (Dako, Carpinteria, CA), and K and )t light chains (Neomarkers, Fremont, CA).
RNA In Situ Hybridization In situ hybridization for K and X light chain mRNA and EBER-1 RNA was performed using a modification of the method of Weiss et a116,17 as described by Aguilera et aP s and biotinylated oligonucleotide probes for K (4 oligonucleotides) and X (6 oligonucleotides) mRNA and the EBER-1 probe for EBV-RNA (30 base digoxigenin-labeled oligonucleotide; Research Genetics, Huntsville, AL). Gcnotypic Studies Bone marrow aspirate suspended in ethylinediaminetetraacetic acid and fresh tissue from the spleen and liver biopsy were obtained. Methods used for Southern blot analysis (SBA) and DNA probes for the immunoglobulin hea W chain gene
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j o i n i n g region (JH) and EBV terminal repeat region have been described previously.19 The T-cell receptor [3-chain probe was described by Weiss et al. 2° RESULTS HistopathologT/Immunophenotype Retrospective review of the initial bone marrow biopsy, interpreted elsewhere as ITP, showed scattered lymphoid aggregates with variable numbers of admixed histiocytes and some with rare R-S-like cells (Table 1). Most lymphocytes were CD3 and CD8 positive. The R-S-like cells were CD15, CD30, and EBV-positive. Histological sections from the spleen showed an extensive red pulp infiltrate and small white pulp nodules composed of small lymphocytes, plasma cells, histiocytes (some showing erythrophagocytosis), and conspicuous large mononuclear and multinucleated cells with prominent nucleoli resembling R-S cells and R-S variants (Fig 1A). Small foci of more numerous plasma ceils were also present. Immunohistochemical stains supported a T-cell origin for the large cells (Fig 1B, Table 1). In situ hybridization studies showed small clusters of K light chain class restricted plasma cells (Fig 1C). The vast majority of R-S-like cells, occasional plasma cells, and variably sized lymphoid ceils were EBV-positive. The liver biopsy performed at the time of splenectomy showed scattered atypical lymphoid cells within the sinusoids but was not considered diagnostic of a PTLD. A subsequent liver biopsy, 3 months later, showed portal tracts expanded by a lymphoplasmacytic infiltrate with numerous EBV-positive plasma cells that showed a marked K predominance. The repeat bone marrow biopsy from September 1994 showed focal relatively homogeneous aggregates of plasma cells throughout the medullary cavity (Fig 2). Some plasma cell aggregates had many admixed lymphocytes, and some were associated with granulomas. The plasma cells showed a marked K predominance. Moderate numbers of small CD3positive lymphocytes, mostly associated with the plasma cell
TABLE 1. H i s t o p a t h o l o g y a n d I m m u n o p h e n o t y p i c Results BM* (6/94) Histopathology R-S-like cells Plasma cells Antigen Total/R-S like cells§ Kappa Lambda LCA CD20 (L26) CD79a CD3 CD8 CD15(LeuM1) CD30(BerH2) EMA EBER-ISH
Spleen (7/94)
Liver (9-10/94)
BM (9/94)
+* +
+ + +t + +j-
+ +++
+++
+/+/+++/+ -/ND +++/+++/+++/+ -/++ -+/-4+/++
++/+/+++/-+ +/+/+++/+++ +++/+/+ +++/+++ +/+ +/+++
+++/+/ND +/ND ++/ND -/++/+ ND ++
+++/+/ND +/ND ++/+ ++/+++/ND ND +++
FIGURE 1. T-cell predominant PTLD with Hodgkin-like features in spleen. (A) Note the pleomorphic diffuse infiltrate including large binucleated and multinucleated cells resembling R-S cells, (B) These ceils stained with the polyclonaI CD3 antibody. (C) In situ hybridization stains showed small foci of K positive plasma cells (left) in areas in which the X stain was negative (right). ([A] Hematoxylin and eosin [H&E] stain. [B] CD3 immunoperoxidase stain with hematoxylin counterstain, [C) in situ hybridization stain for K and X with hematoxylin counterstain).
Abbreviations: R-S, Reed-Sternberg; LCA, leukocyte common antigen; EMA, epithelial membrane antigen; EBER, Epstein Barr virus encoded RNA;ISH, in situ hybridization. *+ = scattered (up to a few seen on scanning). t + + = moderate (easilyfound). ~.+ + + = numerous (present in every field). §Results shown separately for total lymphocyte population and R-S-like ceils.
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aggregates, were seen. Rare R-S--like cells were also CD3positive. Both lymphocytes and plasma cells showed positivity for EBV. Genotypic Studies The genotypic studies, as summarized in Table 2, showed clonal TCR-[3 chain gene rearrangement in the spleen and
CASE STUDIES
FIGURE 2. Plasma cell rich PTLD involving the bone marrow (September 1994). Note the numerous plasma cells replacing portions of the medullary space. These cells showed monetypic ~ light chain expression. There are some admixed histiocytes, (H&E stain.) subsequent bone marrow biopsy from September 1994 (Fig 3). The immunoglobulin heavy chain gene was clonally rearranged in the bone marrow whereas the spleen and liver showed only germline bands. EBV terminal repeat analysis showed three clonal bands in the spleen and two or three clonal bands in the liver and bone marrow. DISCUSSION The case reported here broadens the concept of how PTLD can show a histological, phenotypic, and genotypic spectrum of disease within an individual patient and how PTLD can evolve over time. The patient had an EBV associated T-cell PTLD at some sites and a plasma cell rich EBV-associated B-cell PTLD that was most prominent at other sites. Ancillary studies however supported the coexistence of both PTLD at most sites. The T-cell PTLD with some Hodgkin'slike features was most apparent in the initial bone marrow biopsy and in the spleen. The increasing predominance of the B-cell clonal proliferation seen in the subsequent marrow and liver biopsy specimens occurred after the reduction in immunosuppression and the administration of antiviral agents. The initial T-cell PTLD, which bore some resemblance to Hodgkin's disease, seems similar to some of the other T-cell PTLDs that have been reported. Fourteen percent of all PTLD have been reported to be of T-cell origin.8 When tested, most have shown clonal T-cell receptor gene rearrangement (8 of 9 cases s) similar to the current case. EBV positivity, as seen here, has been reported in at least seven other T-cell PTLD. 8,12,2I The reported cases of T-cell PTLD show a histopathologic spectrum including pleomorphic and anaplastic large cell lymphomas, one of which had CD30 positivity and splenic
TABLE 2. Genotype
BM (6/94)
TCR-[3 JH EBV (terminal repeat analysis)
ND ND ND
involvement.8 The latter case was not clearly associated with EBV. The plasma cell rich B-cell P ~ D principally involved the allograft liver and bone marrow specimens obtained after the patient's immunosuppression had been reduced. These lesions show architectural destruction and are predominantly composed of plasma cells and plasmacytoid cells with few transformed cells, is It is of interest that although the B-cell clone and numerous plasma cells predominated only in the later PTLD, a small K restricted plasma cell component was present in the splenectomy specimen with the T-cell PTLD. Although not seen with the SBA using the JH probe, the EBV terminal repeat analysis supports the observation that the major clone present in the later marrow and liver biopsy specimens is present as a minor clone in the spleen. Conversely, the later marrow which histologically and phenotypically has a predominant B-cell clone, also shows evidence of an identical T-cell clone. It is uncertain why the major EBV band present in the spleen is hardly detected in the later marrow because the latter includes a component of the original T-cell PTLD. These morphological, immunophenotypic, and genotypic results support the presence of a multiclonal PTLD in which the more anaplastic T-cell component showed an apparent significant response to decreasing the patient's immunosuppression; however, the more indolent appearing clonal plasma cell rich component showed an apparent growth advantage. The patient ultimately required conventional chemotherapy for control of his PTLD. The current case shares some features with that of a renal transplant recipient who developed a cutaneous T-cell lymphoma followed by a nodal lymphoma containing both B- and T-cell clones and EBV.22 The phenotype a n d / o r genotype of the EBV-positive cells was not clear, and EBV was not shown in the cutaneous T-cell PTLD. A second renal transplant recipient developed a scalp-based EBV-positive B-cell PTLD (T-cell clonality not investigated) and a subsequent T-cell clonal population was shown in the peripheral blood. 12It is unknown if EBVwas present in the T-cell clone. A third renal transplant recipient showed allograft involvement by a phenotypically B-cell PTLD with genotypic evidence of both immunoglobulin and T-cell receptor gene rearrangement. 21 This patient's PTLD, however, did not show two histologically distinct lesions and there is no evidence that the gene rearrangements were in two distinct clones. Although seemingly unique, the existence of a PTLD such as reported here is not surprising given the many previous observations that individual patients may have distinct B-cell clones at multiple or even single sites. 1,I5,19 Furthermore, PTLDs are known to relapse sometimes with morphologically a n d / o r clonally distinct disease. 5 The current case, however, highlights the need to consider the presence of simultaneous or subsequent B- and T-cell clones and the need to biopsy different sites of disease particularly when patients
G e n o t y p i c Studies
Spleen (7/94)
Liver (9-10/94)
BM (9/94)
Clonal & polyclonal pattern* ND Clonal & polyclonal pattern* Germline Germline Clonal (faint) 1 major & 2 minor clonal 1 major & 1 minor clonal band]- 1 major & 2 minor clonal bands]bandst
Abbreviations: TCR-13,T-cellreceptor beta chain gene;JH, immunoglobinheavy chain gene,joining region; EBV,Epstein Barr virus. *The TCR-[3clone in the spleen and the bone marrow show similar electrophoretic mobility suggesting that they represent the same clone at both sites. ]-The two minor EBV clones in the spleen are in similar positions as the major and minor clones in the liver and bone marrow.
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1 2 3
EcoRV
Jn BglII
EBV-Terminus BamHI
FIGURE 3. Genotypic results using SBA. The arrows at the far left indicate the bands representing common clonal rearrangement of the TCR-I~ chain gene in the spleen and bone marrow (September 1994). Immunoglobulin heavy chain rearrangement was identified in the marrow (September 1994), but not the spleen (arrow in middle panel). EBV terminal repeat analysis showed major (#1 in panel on right) and 2 minor clonal bands (#2 and #3) in the spleen. The liver and bone marrow e a c h show a major and a minor clonal band although a faint band in position 1 may also be present in marrow. Note the similar location of one minor EBV clonal band at all three sites (#3). The major EBV clonai bands of the liver and bone marrow are also in a similar position to a minor clonal band in the spleen (#2).
do not respond to decreased immunosuppression or when disease recurs. Finally, this patient was initially diagnosed as having idiopathic thrombocytopenic purpura even after a bone marrow biopsy. Thrombocytopenic purpura with increased marrow megakaryocytes can follow EBV infectionZS; however, this case proved to be a PTLD. It therefore emphasizes the need to always maintain a high degree of suspicion for PTLD in patients after transplantation. REFERENCES 1. Nalesnik MA, Jaffe R, Starzl TE, et ah The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine Aprednisone immunosuppression. AmJ Pathol 133:173-92, 1988 2. Frizzera G, Hanto DW, Gajl-Peczalska KJ, et al: Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients. Cancer Res 41:4262-4279, 1981
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3. Kxlowles DM, Cesarman E, Chadburn A, et al: Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood 85:552-565, 1995 4. Swerdlow SH: Classification of the posttransplant lymphoproliferative disorders: From the past to the present. Semin Diagn Pathol 14:2-7, 1997 5. Wu T-T, Swerdlow 8H, Locker J, et al: Recurrent Epstein-Barr-Virus associated lesions in organ transplant recipients. HUM PATHOL27:157-64, 1996 6. Waller EK, Ziemianska M, Bangs CD, et al: Characterization of posttransplant lymphomas that express T-cell-associated markers: Immunophenotypes, molecular genetics, cytogenetics, and heterotransplantation in severe combined immunodeficient mice. Blood 82:247-61, 1993 7. Ross CW, Schnitzer B, Sheldon S, et al: Gamma/Delta T-cen posttransplantation lymphoproliferative disorder primarily in the spleen. Am J Clin Pathol 102:310-315, 1994 8. vanGorp J, Doornewaard H, Verdonck LF, et al: Posttransplant T-cell lymphoma. Report of three cases and a review of the literature. Caiacer 73:3064-3072, 1994 9. Kaplan MA, JacobsonJO, FerryJA, et al: T-cell lymphoma of the vulva in
CASE STUDIES a renal allograft recipient with associated henmphagocytosis. AmJ Snrg Pathol 17:842-849, 1993 10. Morrison VA, Dunn DL, ManivelJC, et al: Clinical characteristics of post-transplant lymphoproliferative disorders. AmJ Med 97:14-24, 1994 11. Nalesnik MA, Randhawa P, Demetris AJ, et al: Lymphoma resembling Hodgkin disease after posttransplant lymphoproliferative disorder in a liver transplant recipient. Cancer 72:2568-2573, 1993 12. Frankel AH, Tbompson M, Vulliamy T, et al: A T cell clone in association with an Epstein-Barr virus-related B cell lymphoma (Letters to Editor). Transplantation 52:1108-1110, 1991 13. SwerdlowSH: Posttransplant lymphoproliferative disorders: A working classification. Curt Diagn Pathol 4:29-36, 1997 14. SwerdlowSH:Post-transplant lymphoproliferative disorders:Amorpholo,~c, phenotypic and genotypic spectrum of disease. Histopathology 20:373385, 1992 15. Chadburn A, Cesarman E, YiFang L, et al: Molecular genetic analysis demonstrates that multiple posttransplant lymphoproliferative disorders occurring in one anatomic site in a single patient represent distinct primary lymphoid neoplasms. Cancer 75:2747-2756, 1995 16. WeissLM, Movahed LA, Chen Y-Y,et al: Detection ofimmunoglobulin light-chain mRNA in lymphoid tissues using a practical in situ hybridization method. AmJ Patho1137:979-988, 1990
17, Weiss LM, Chen Y-Y,Liu X-F, et al: Epstein-Barr Virus and Hodgkin's Disease. A correlative in sire hybridization and polymerase chain reaction study. AmJ Pathol 139:1259-1265,1991 18. Aguilera NS, Kapadia SB, Nalesnik NLA,et ah Extramednllary plasmacytoma of the head and neck: Use of paraffin sections to assessclonalitywith in situ hybridization, growth fraction, and the presence of Epstein-Barr virus. Mod Pathol 8:503-508, 1995 19. Locker J, Nalesnik M: Molecular genetic analysis of lymphoid tumors arising after organ transplantation. AmJ Pathol 135:977-987, 1989 20. WeissLM, Hu E, Wood GS, et al: Clonal rearrangements of the T-cell receptor gene in mycosis fangoides and dermatopathic lymphadenopathy. N EnglJ Med 313:539-544, 1985 21. Hollingsworth HC, Stetler-StevensonM, Gagneten D, et al: Immunodeficiency-associated malignant lymphoma. Three cases showing genotypic evidence of both 2"-and B-celllineages. AmJ Surg Pathol 18:1092-1101, I994 22. Euvrard S, Noble CP, Nanitakis J, et ah Brief report: Successive occurrence of T-cell and B-cell lymphomas after renal transplantation in a patient with multiple cutaneous squamous-cen carcinomas. N Engl J Med 327:1924-1926, 1992 23. MatsukawaY, Okano M, Ishikawa N, et aI: Severe thrombocytopenic purpura associated with primary Epstein-Barr virus infection. J Infect 29:107109, 1994 (letter)
ENHANCED EXPRESSION OF INTERLEUKIN-6 IN BONE AND SERUM OF METASTATIC RENAL CELL CARCINOMA BERNARD PAULE, MD, DIDIER CLERC, MD, CHRISTIANE RUDANT, COLETTE COULOMBEL, LAURENCE BONHOMME-FAWRE, MD, JEANINE QUILIARD, MD, AND MICHEL BISSON, MD
Interleukin-6 (IL-6) is produced by renal cell carcinoma (RCC) cell lines and primary tumors. Using immunohlstochemlcal staining in two RCC patients with hypercaleemia and high serum levels of free and total II~6, we showed expression of IL-6 in metastatic bone tissue. The role of IL-6 ha hypercalcemia and bone resorption would suggest that bisphosphonates or dexamethasone could be useful as adjuvant therapy for IL-6 dependant bone metastases which fail to respond to interferon alpha (IFN) ol 2a and all traxts retinoic add
(ATRA). HUM PATHOL29:421424. Copyright © 1998 byW.B. Saunders Company Key words: Interleuldn-6, bone metastasis, renal cell carcinoma, immunohistochemistry. Abbreviations: IL-6, Interleukin 6; RCC, renal cell carcinoma; IFN, interferon; ATRA, All trans retinoic acid; sIL-6R, Soluble receptor to IL-6; PBS, Phosphate-buffered saline; PTHrP, Parathyroid hormone related peptide.
I n t e r l e u k i n - 6 (IL-6) is a p l e i o t r o p i c c y t o k i n e s y n t h e t i z e d b y b o t h n o r m a l a n d t u m o r a l cells. 1 I t is p r o d u c e d b y r e n a l cell c a r c i n o m a ( R C C ) c e l l l i n e s 2,3 a n d i n patients with primary RCC, 4 and has been shown to act as a n R C C a u t o c r i n e g r o w t h f a c t o r . 5,6 E l e v a t e d s e r u m l e v e l s o f IL-6 b e f o r e t r e a t m e n t a r e c o r r e l a t e d w i t h shorter survival and lack of response to immunotherapy, v In two patients with advanced stage RCC and h y p e r c a l c e m i a , w e e v a l u a t e d IL-6 e x p r e s s i o n i n b o n e m e t a s t a t i c t i s s u e s a n d s e r u m l e v e l s o f f r e e IL-6 o r IL-6 b o u n d t o its s o l u b l e r e c e p t o r ( s I L - 6 R ) .
L a s e g u e ' s sign, clinical e x a m i n a t i o n was u n r e m a r k a b l e . H e h a d raised e r y t h r o c y t e s e d i m e n t a t i o n rate (ESR) (90 m m 1st h o u r ) , C-reactive p r o t e i n (CRP: 149 m g / L , N L < 112 m g / L ) a n d calcemia (2,90 m m o l / L , NL: 2,25-2,60 m m o l / L ) ; comp l e t e b l o o d count, c r e a t i n e m i a , a n d liver enzymes were n o r m a l . S e r u m i n t a c t p a r a t h y r o i d h o r m o n e (10 p g / m L , NL: 10 to 65 p g / m L ) , 25 h y d r o x y v i t a m i n D (7,2 n g / L , NL: 8 to 30 n g / m L ) were lowered a n d calcitriol (27 p g / m L , NL: 20 to 80 p g / m L ) was n o r m a l . R a d i o g r a p h s disclosed a n o r m a l l u m b a r s p i n e a n d a r i g h t sacral osteolysis. Sacrum, r i g h t t r o c h a n t e r i c , a n d ilio-ischiatic h o t spots were revealed o n t e c h n e t i u m 9 9 m (99mWc) skeletal scintigraphy. Lumbo-sacral, a b d o m i n a l a n d t h o r a c i c c o m p u t e r i z e d t o m o g r a p h i c studies respectively docum e n t e d (1) a large (6,5 c m × 7 c m × 4,5 cm) lyric L5 a n d $1 v e r t e b r a l a n d sacral lesion i n v a d i n g t h e spinal canal, compressi n g t h e t h e c a l sac a n d a d h e r i n g to t h e iliac vessels; (2) a r e n a l tissue mass w h i c h was e n h a n c e d after injection; (3) five r i g h t p u l m o n a r y i n f r a c e n t i m e t r i c n o d u l e s a n d a 6 5 m m large subp l e u r a l mass in t h e p o s t e r o b a s a l s e g m e n t o f t h e left lower lobe. A sacral biopsy was p e r f o r m e d . P a t h o l o g i c a l study c o n c l u d e d to a p o o r l y d i f f e r e n t i a t e d a d e n o c a r c i n o m a with clear cells in favor o f a r e n a l origin. H y p e r c a l c e m i a a n d p a i n resolved w h e n t r e a t e d by p a m i d r o n a t e infusions, oral corticosteroids, a n d m o r p h i n e . S u b c u t a n e o u s i n t e r f e r o n (IFN) c~2a (Roferon-A, H o f f m a n n - R o c h e , France, 18 million units 3 days a week) a n d oral all trans r e t i n o i c acid (ATRA, H o f f m a n n Roche, France, 100 m g / m 2 / d a y given in t h r e e equally divided doses) were i n s t a u r e d o n D e c e m b e r 13th as described, s O n e m o n t h later, r e c r u d e s c e n t p a i n c a u s e d by sacral t u m o r progression led to e m b o l i z a t i o n t h e n L5 v e r t e b r a l a n d s a c r u m r a d i o t h e r a p y (45 Gy, five f r a c t i o n s / w e e k , 2 G y / f r a c t i o n ) .
PATIENTS AND M E T H O D S :
Case 1 A 55-year-old m a n was a d m i t t e d in D e c e m b e r 1993 with a 2 - m o n t h history o f r i g h t S1 sciatica a n d r e c e n t loss in weight o f 8 kilograms. A p a r t f r o m a l u m b a r stiffness a n d a r i g h t From the Service de Rhumatologie, H6pital de Bic~tre, Le Kremlin-Bic6tre, France; the UFR Biom6dicale UPR 37, R6gulation de l'expression des g~nes, Paris, France; the Service de Pharmacie, D6partement de Pharmacologie, H6pital Paul Brousse, Villejuif, France; and the Service d'Anatomo-pathologie, H6pital de Bic&re, Le Kremlin-BicStre, France. Address correspondence and reprint requests to Bernard Paule, MD, Service de Rhumatologie, H6pital de BicStre, 78 rue dn G6n~ral Leclerc, 94 275 Le Kremlin Bic6tre, France. Copyright © 1998 by W.B. Sannders Company 0046-8177/98/2904-000X$8.00/0
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