Lymphomatoid papulosis and progression to T cell lymphoma: An immunophenotypic and genotypic analysis

Lymphomatoid papulosis and progression to T cell lymphoma: An immunophenotypic and genotypic analysis Douglas S. Harrington, MD,a Suzanne W. Braddock, MD,b Kathleen S. Blocher, MD,b Dennis D. Weisenburger," Warren Sanger, Phf),? and James O. Armitage, MDb

Omaha, Nebraska A 37-year-oldwhite man had untreated lymphomatoid papulosis for 12 yearsbefore a submandibular T cellimmunoblasticlymphoma developed. A genetic abnormality, composedof extra chromosomalmaterial attached to the short arm of chromosome 9, wasdetectedin the lymphoma tissue but not in the skin. The lymphomatoidpapulosis skinlesions did not manifestclonalT cellreceptor generearrangements, but thesubmandibularlymphoma tissuewas clonaland of T cell lineage. The patient'slymphomaresponded wellto combination chemotherapy, but the lymphomatoid papulosis remains active. (J AM ACAD DERMATOL 1989;21 :951-7.) MATERIAL AND METHODS

Lymphomatoid papulosis is characterized by crops of papules and papulonodules that may ulcerate and resolve spontaneously, leaving hypopigmented or hyperpigmented atrophic scars. I, 2 The lesions typically involve the trunk and extremities, may be in various stages of evolution, and last from 3 to 8 weeks. 1, 2 The histologically malignant appearance of the lesions contrasts with the apparent benign clinical behavior of lymphomatoid papulosis.l-? Results of early studies suggested that lymphomatoid papulosis did not threaten the patient's health, but those of subsequent reports have demonstrated lymphomatoid papulosis in association with mycosis fungoides, Hodgkin's disease, and nonHodgkin's lymphoma."? Lymphomas will eventually developin approximately 10%to 19%of patients with lymphomatoid papulosis.s 7,10 We describe a patient with untreated lymphomatoid papulosis of 12 years' duration in whom a T cell immunoblastic lymphoma subsequently developed. We applied molecular biologic and immunophenotypic techniques to assess the relation of the lesions.

From the Departments of Pathology and Microbiology," Internal Medicine," and Human Genetics," University of Nebraska Medical Center. Accepted for publication Dec. 5, 1988. Reprint requests: Douglas S. Harrington, MD, Assistant Professor, Department of Pathology and Microbiology, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, NE 68105-1065.

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Lymph node and skin biopsy protocol Separate portionsof eachbiopsy specimen werefixed in 10%buffered formalin for24 hours andB5_fixative before theywereembedded in paraffin andstainedwithhematoxylin-eosin stain. A portion of each specimen was snap-frozen in Tissue-Tek Optimum Cutting Temperature (OCT) compound (Miles Scientific, Naperville, lli.) by immersion of the specimen in liquidnitrogen ora slurry of N-methyl-butanol and dry ice. The tissue was then stored in airtight bags at -70 0 C and subsequently stained with a panelof monoclonal T andB cellmarkers within48hours.Portions oftissuealsowere submittedfor flow cytometric, cytogenetic, and gene probestudies.

Immunohistologic surface marker analysis Cryostat-sectioned specimens (6 /-Lm) were cut and placed on albumin-coated slides. Sections were warmed to room temperature and fixed in acetone for 5 minutes. They were then washed with phosphate-buffered saline solution(0.1 mol/L, pH 7.4) and, finally, incubated with selected monoclonal antibodies or with polyvalent rabbit antibodies. After thesections were washed three times in phosphate-buffered salinesolution, bound antibody was demonstrated with the use of an avidin-biotin immunohistochemical procedure (Vectastain, Vector Laboratories, Burlingame, Calif.). Monoclonal antibodies used in this study were Bl, B2, and B4 (Coulter Immunology, Hialeah, Fla.); BA1 and BA2, (Hybritech, San Diego, Calif.); Leu-I through Leu-9, Leu-14, anti-Tac, and anti-human lymphocyte antigen-DR (anti-HLA-DR) (Becton-Dickinson, Sunnyvale, Calif.); and OKT9 (Ortho Pharmaceuticals, Raritan, N.J.). Polyclonal anti-

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Harrington et ai.

Fig. 1. A, Low-power view of right submandibular lymph node shows infiltration of adjacent soft tissue and muscle fibers by predominantly large cell infiltrate. (Hematoxylin-eosin stain; X 100.) B, High-power view shows pleomorphic, large, immunoblastic lymphoid cells and scattered, atypical, small lymphoid cells. (Hematoxylin-eosin stain; X 400.)

bodies were anti-kappa, anti-lambda, Ki-I, and antiimmunoglobulin heavy chains (IgG, IgA, IgM, IgD) (Dako, Santa Barbara, Calif.).

Cytogenetics methods Lymph node, cutaneous biopsy, bone marrow, and peripheral blood specimens were processed for cytogenetic studies within 1 hour of biopsy according to a standard protocoL 11 Briefly, the lymph node and skin tissue were minced and placed directly into Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Madison, Wis.), supplemented with 20% fetal bovine serum and gentamicin. The disaggregated tissue was then cultured for 24 hours, exposed to colcemid for 2 hours, and harvested for cytogenetic studies.

Gene probe studies Diagnostically indicated exeisional or punch biopsy specimens were obtained after informed consent and processed according to the outlined protocol. High-molecular-weight DNA was extracted from snap-frozen samples by proteinase-K digestion, phenol-chloroform extraction, and ethanol precipitation with the use of standard proceduree.l- 13 DNA (10 ,ug) was digested with restriction enzymes (EcoRI, HindIII, BamHI) according to the manufacturer's directions (New England Biolabs, Bever-

ly, Mass.). Restriction-cut DNA was then size fractionated by electrophoresis in 0.8% agarose gels and transferred to nylon filters with the use of the Southern method. 12 A human T cell antigen receptor beta chain Pstl 0.7 Kb gene probe and an immunoglobulin heavy chain gene Sau3A 2.5 Kb DNA probe were labeled with 32p nucleotides and hybridized for 12 to 18 hours. 14. 15 The filters were then washed to stringency and exposed for 48 to 72 hoursat-70° ConKodakX-OmatARfilm with the use of intensifying screens. Control DNA included placenta (gerrnline), MOLT-4 cell line (T cell receptor gene rearrangement-positive), and Raji cell line (immunoglobulin gene rearrangement-positive).

CASE REPORT A 37-year-old white man was referred to the University of Nebraska Medical Center in September 1984'with a lO-day history of a mass under the right side of his jaw. He had a 12-year history of self-healing recurrent cutaneous lesions on the extremities and trunk. Physical examination revealed a right submandibular nodule, 2 X 2 em, that was not fixed to the overlying skin but was adjacent to a hypopigmented scar. Lymphadenopathy and hepatosplenomegaly were absent. Routine laboratory findings were normal. Radiographs of the

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Lymphomatoid papulosis and progression to T eel/ lymp homa 953

chest, maxilla, and mandible, a bipedallymphangiogram, and a computed tomographic scan of the abdomen and pelvis showed normal findings. Bone marrow aspiration and biopsy specimens were negative for malignant lymphoma. Examination of the skin revealed approximately 40 erythematous, scaly, firm, cutaneous nodules, 0.5 to 1 em, on the flexor and extensor surfaces of the arms and legs. The lesions were in various states of activity. A diagnosis of immunoblastic T cell lymphoma was made on the basis of histologic features and surface markers, and the patient subsequently received total skin electron beam radiation and five courses of six-drug combination chemotherapy, including cyclophosphamide, doxorubicin (Adriamycin), procarbazine, bleomycin, vincristine (Oncovin) , and prednisone (CAP-BOP).ll No new skin lesions were noted after the first course of CAPBOP, and the right submandibular mass resolved completely. The patient now has approximately 50 lymphomatoid papulosis lesions scattered on the trunk and arms in varying states of activity or regression. There is no evidence of recurrent lymphoma 4 years after diagno-

sis. RESULTS

Histopathologic findings Examination of a biopsy specimen of the submandibular mass revealed an immunoblastic large cell lymphoma that extended into the surrounding soft tissue. There was no evidence of lymphomatoid papulosis in continuity. The mass was composed of pleomorphic, large lymphoid cellswith round nuclei, vesicular chromatin, a thin nuclear membrane, small nucleoli, and moderate to abundant pale, eosinophilic cytoplasm with well-demarcated borders (Fig. 1, A). Scattered atypical small lymphoid cells with markedly irregular nuclear contours were admixed. Transitional lymphoid cells, mitotic figures, and scattered eosinophils also were noted (Fig. 1, B). Cytomorphometric analysis showed that the large cells represented 60% to 80% of the lymphoid cells. The tumor extensively infiltrated through the lymph node capsule into the adjacent skeletal muscle and adipose tissue. Biopsy specimens of skin lesions obtained at the time of lymphoma diagnosis (1984) and during follow-up (1984 to 1986) were similarin appearance to those obtained in 1977 and 1980. The predominant feature was a dense interstitial cellular infiltrate that filled the papillary and reticular dermis (Fig. 2, A). The infiltrate was composed of a mixture of atypical small and, lesscommonly, large lymphoid cells,with scattered benign-appearing histiocytes admixed

(Fig. 2, B). The small lymphoid cells predominated and were characterized by markedly irregular nuclear contours, coarsely clumped chromatin, inconspicuous nucleoli, and scant cytoplasm. The large lymphoid cells had round or slightly irregular nuclei, vesicular chromatin, prominent nucleoli, and moderate to abundant amounts of cytoplasm. Scattered rare large multinucleate Reed-Sternberg-like cells were evident. Mitotic figures were frequent. The epidermis showed variable degrees of spongiosis, necrosis, ulceration, and crusting. The papillary dermis was edematous, and small blood vessels appeared hyperplastic. Examination of four biopsy specimens taken at various intervals during therapy showed a similar but less intense, predominantly perivascular, infiltrate with slight exocytosis.

Immunohistochemical findings The lymphoma cells were activated helper T cells because they were Leu-5 (CD2), Leu-4 (CD3), Leu-3 (CD4), Ki-l (CD30), HLA-DR, OKT9, and Tac (CD25)-positive. Reactions with Leu-l (CD5), Leu-9 (CD7), and ' Leu-2 (CD8) were negative. Leu-6 (CDl )-positive Langerhans cells were lacking. In the skin lesions, the vast majority of small and large lymphoid cells were activated helper T cells because they were Leu-l (CDS), Leu-4 (CD3), Leu-5 (CD2), Leu-3 (CD4), OKT9, and Tac (CD25 )-positive. Scattered Leu- 2 (CD8)-positive cytotoxic/suppressor T cells and Leu-6 (CDI)-positive Langerhans cells also were present. The large lymphoid cells also were Ki-I (CD30)-positive. Only the small cells Were Leu-9 (CD7)-positive.

Cytogenetics G-banded chromosome studies of lymphoma tissue revealed a clonal translocation of unidentifiable material to the short arm of chromosome 9; t(9:?) (p22-23;?) (Fig. 3). R-banding techniques failed to identify the translocated material further. No normal cells were identified. Cultured cutaneous tissue failed to yield mitotic cells, but bone marrow and phytohemagglutinin-stimulated peripheral blood lymphocytes had normal karyotypes.

Gene probe analysis A monoclonal population of T cells was identified in the lymphoma biopsy specimen with the use of Southern analysis with the T cell antigen receptor beta-chain probe, confirming the histologic diagno-

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Fig. 2. A. Low-powerviewshows densedermal infiltrate in skin. (Hematoxylin-eosin stain; X 40.) B, High-power view shows skin infiltrate to be mixture of atypical small and, less commonly, large lymphoid cells. (Hematoxylin-eosin stain; X 400.) sis of lymphoma (Fig. 4). The immunoglobulin heavy-chain genes (B cells) were germline (nonclonal) (analysis not shown). No monoclonal populations of T cells were identified by gene rearrangement studies in the two skin lesion biopsy specimens obtained at the time of lymphoma diagnosis or in two subsequent biopsy specimens obtained after chemotherapy (Fig. 4). The immunoglobulin loci were germline,

DISCUSSION

We have described a patient with a 12-year history of lymphomatoid papulosis in whom a T cell lymphoma subsequently developed to explore the relation between the two lesions. Our analysis indicates the T cell lymphoma probably resulted from progression of the lymphomatoid papulosis.

Histologic similarities were evident between the lymphomatoid papulosis lesions and the submandibular tumor. Rare large immunoblast-like cells were observed in the lymphomatoid papulosis lesions, which resembled those in the submandibular tumor. The large cells predominated in the submandibular lesion (60% to 80%) but were less conspicuous in the lymphomatoid papulosis skin lesions (8%). The immunophenotypes of the two lesions were similar with the exception of Leu-I (CD5), which was expressed on the large cells of the lymphomatoid papulosis lesions, but not on those of the submandibular lesion. This does not rule out transformation of the lymphomatoid papulosis to malignant lymphoma because T cell lymphomas often express aberrant phenotypes in which pan T cell markers have been lost.16

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Lymphomatoid papulosis and progression to T cell lymphoma 955 2

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Fig. 3. Extra chromosomal material translocated to short ann of chromosome 9 (arrow).

Analysis of clonality with the use of gene rearrangement studies revealed that the submandibular mass had a clonal population of T cells, but the lymphomatoid papulosis lesion was not demonstrably clonal. This does not rule out a clonal population in the lymphomatoid papulosis lesions, because the neoplastic population could have been below the level of detection of our DNA analysis system (1% to 2%). A cytomorphometric differential, however, revealed that the large cells in the lymphomatoid papulosis lesions represented approximately 8% of the lymphoid cells, whereas those in the submandibular lesion represented 60% to 80%. It seems reasonable to assume that a clonal population, if it was represented by the large cells, would be detected by our system. Clonal populations of T cells have been identified in a majority of patients with lymphomatoid papulosis who have been studied.?: 17 There were significant differences in the study populations, however. Of the six patients analyzed by Weiss et al.,17 five had clonal T cell receptor gene rearrangements and the sixth had a polyclonal rearrangement. Three of five patients who manifested clonal rearrangements of skin lesions had lymphoma (two, mycosis fungoides; one, large cell) that preceded the lymphomatoid papulosis, and they may actually have had cutaneous involvement masquerading as lymphomatoid papulosis. Two patients did have lymphomatoid papulosis, and clonal populations of T cells were identified. The sixth patient's lesions were not monoclonal. Other studies have shown that more than 50% of cases of lymphomatoid papulosis appear to

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Fig. 4. Southern analysis results. A, Submandibular lymph node shows two rearranged bands (arrowheads), confirming clonal T cell population. Band C, Skin lesion biopsy specimens at time oflymphoma diagnosis (B) and 1 year after chemotherapy and electron beam therapy (C) show only germline bands (dashes).

be clonal.v 17 Our patient had lymphomatoid papulosis that preceded the lymphoma for 12 years. The lymphomatoid papulosis and lymphoma have responded independently to therapy. The submandibular mass resolved with chemotherapy after

956 Harrington et al. biopsy, whereas the lymphomatoid papulosis has remained active after chemotherapy and electron beam therapy. This may reflect the nonaggressive nature of the lymphomatoid papulosis as opposed to the more aggressive nature of the lymphoma. Lowgrade lymphoproliferations are less susceptible to therapy as a result of low proliferative indexes. IS Patients with lymphomatoid papulosis have an increased risk of Iymphoma.v 7,10 but whether lymphomatoid papulosis is premalignant is still not clear. Lymphomatoid papulosis may be a clonal T cell disease held in check by the host immune system that becomes more aggressive and progresses to overt lymphoma when the host immune response is inadequate to control the process.'? Nonclonal or minimally clonal skin lesions, however, can apparently precede lymphoma, which appears to have been the case in our patient. Because only clonal T cell rearrangements have been detected in lymphomatoid papulosis, the incidence of T cell lymphoma in lymphomatoid papulosis patients should be greater if progression occurs. Lymphomatoid papulosis, however, can precede or occur after successful treatment of other types of lymphoma that are not ofT cell lineage, including Hodgkin's disease and B celllymphomas.S 17 The Reed-Sternberg cells of Hodgkin's disease, however, have an immunophenotype similar to that of the large cells oflymphomatoid papulosis.'? Lymphomatoid papulosis probably reflects an immune defect in the host,2o As such it may begin as an atypical hyperplasia of skin-associated lymphoid tissue that can progress to an oligoclonal or monoclonal lymphoproliferation. In this setting, each tumor can have a unique clone unless dissemination occurs. Transformation to a more aggressive lymphoproliferation may be the result of further cytogenetic events with subsequent oncogene activation that overwhelms the already compromised immune surveillance system. The prototype for this type of lymphoma genesis is the transplant-associated lymphomas in which this type of progression has been documented/" Polyclonal B cell proliferations have been shown to progress to oligoclonal and, finally, monoclonal populations that regress when immunosuppression is decreased or removed." If a further cytogenetic event (i.e., a 14:18 translocation) develops, however, regression does not OCCUr. 2 1 Because immune-deficient states are associated with an increased riskoflymphoma, particularly B celllym-

J ournal of the American Academy of Dermatology

phoma, this would account for the association of lymphomatoid papulosis with non-T cell lymphomas.I' This model would predict that each lesion of lymphomatoid papulosis could have a unique clone of cells if the tumor arose by a singular event in that lesion. Indeed, some lymphomatoid papulosis tumors have unique clones, whereas others share a common banding pattern of c1onality. 9,17 The associated immune defect would also account for the increased incidence of other types of lymphoma in lymphomatoid papulosis. Clonal T cell populations below the threshold of detection by Southern analysis could exist from inception in lymphomatoid papulosis. With time, these clones expand to detectable levels. Chronic lymphocytic leukemia is a monoclonal B celllymphoma/leukemia that has a parallel pattern of behavior.P It can exist for years with little effect on the patient and then suddenly transform into a large cell lymphoma or prolymphocytic leukemia with more aggressive behavior.P As in lymphomatoid papulosis, the events that result in transformation are not well characterized.P The submandibular tumor had a translocation of unknown material to the short arm of chromosome 9 in the region of bands 22 to 23. Although no oncogenes are located in this area, the interferon genes and a locus associated with acute lymphocytic leukemias are (McKusick VA, unpublished observation). An oncogene may possibly have been contained on the-material translocated to this area that could have been activated subsequently; this is speculative without identification of the source of the extra material. Thus far, an insufficient number of patients have been analyzed. Prospective studies are needed that analyze the immune status of patients with lymphomatoid papulosis and uniformly classify and follow up these patients from the inception of the disease to the development of lymphoma should it occur. Clonality studies should address whether individual lesions show unique clonal patterns or whether all lesions contain the same clone. Studies also should distinguish those patients in whom lymphomatoid papulosis lesionsdevelop afterlymphoma from those patients with lymphomatoid papulosis alone or preceding lymphoma. The prognostic significance of immunophenotype and clonality in lymphomatoid papulosis will be defined by longterm follow-up.

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Lymphomatoid papulosis and progression to T cell lymphoma 957

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13. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning. ColdSpring Harbor, New York: Cold Spring Harbor Laboratory Publications, 1982:459-62. 14. FeinbergAP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984;137:266-7. 15. Knowles DM, Pelicci PG, Dalla-Favera R. T-cell receptor beta chain gene rearrangements: geneticmarkers of T-cell lineages and clonality. Hum PathoI1986;17:546-51. 16. Picker LJ, Weiss LM, Mederios LJ, et al. Immunophenotypiccriteria forthe diagnosisof non-Hodgkin'slymphoma. Am J PathoI1987;128:181-201. 17. WeissLM, Wood GS, Trela M, et al. Clonal T-cell populations in lymphomatoid papulosis: evidence of a lymphoproliferative origin for a clinically benign disease. N Engl J Moo 1986;315:475-9. 18. Rosenberg SA. Current conceptsin cancer: non-Hodgkin's lymphoma: selectionof treatment on the basis of histologic type. N Engl J Med 1979;301:924-8. 19. Kadin M, Nasu K, Sako D, et al. Lymphomatoid papulosis: a cutaneousproliferation of activated helper T-cells expressing Hodgkin's disease-associated antigens. Am] PathoI1985;119:315-25. 20. LedermanJS, Sober AJ, Harrist TJ, et al. Lymphomatoid papulosis following Hodgkin's disease. J AM ACAD DERMATOL 1987;16:331-5. 21. ClearlyML, Dorfman RF, Sklar J. Failure in immunological control of the virus infection: post-transplant lymphomas. In: Epstein MA, Achong BG, eds.The Epstein-Barr virus: recent advances. London:William Heinemann Medical Books, 1986:164-77. 22. Harousseau J, Flandrin G, Brouet J, et aI. Malignant lymphoma supervening in chronic lymphocytic leukemia and related disorders. Cancer 1981;1302-8.