Site-dependent phenotypic heterogeneity in peripheral T-cell lymphoma

Ci\SE STUDIES 22. Epstein AL, Mader R], Winter ]1'\, et al: Two new monoclonal anti· bodies (L1'\-I. L1'\-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B-lymphoc)"tes and derh'ed tumors.] ImmunoI133:1028, 1981 23. Bostwick DC, ~[ann RB: ~[alignant lymphomas inmlving the prostate: a study of 13 cases. Cancer 56:2932, 1985

20. lIsu S·~[, Raine L, Fanger H: A comparative study of the peroxidase antiperoxidase method and an avidin-biotin complex method for studying pol)"peptide hormones with radioimmunoassay antibodies. Am] Clin Pathol 75:734, 1981 21. Sheibani K, Tubbs RR: Enzyme immunohistochemistry: technical aspects. Semin Diagn Pathol 1:235, 1984

SITE·DEPENDENT PHENOTYPIC HETEROGENEITY IN PERIPHERAL T·CELL LYMPHOMA RICARDO E. DUQUE. MD, RICARDO BERTRAM SCHl\ITZER, l\ID

V.

LLOYD, PHD,

l\1D, Jom; T.

HEADIl\GT01'\. MD, Al\D

MATERIALS AND METHODS

Peripheral T-celllynpllOmas constitute a heterogeneous populatioll ofposllhynic T-cell malignancies. Characteristically, they present a varied phenot)pie expressioll, which call be helpful ill establishillg the diagnosis. A case of a peripheral T-cell lymphoma ill a 76)'tar-old mall is described. The maligllant cells ill the skill and bOlle marrow were of the T4 (helper/indueel) phenotype, yet they did not express pall-T-cell antigens, such as TIl, orfunctional E roselles. III a biopsy specimell from a lymph node, however, the malignant cells had a helper/illducer phenotype and also expressed the pall-Tcell antigens TIl alld Leu-5. Additionall)', the malignant cells from the l)'lnph II ode formed E rosel/es. This study demonstrates the phellotypic heterogeneity of malignant T cells, which appears to be site-dependent. HUM PATIlOL 17:967-970,1986. Peripheral T-cell lymphoma, a term originally proposed by Waldron et aLI identifies all nonlymphoblastic Tcell lymphomas exclusive of the mycosis fungoides/Sezary syndrome. 2 Peripheral T-cell lymphomas have a diffuse growth pattern and are cytomorphologically heterogeneous. 2 •3 Furthermore, careful studies ha\'e demonstrated that these lymphomas exhibit considerable immunologic diversity. In fact, in a high percentage of cases, the malignant T cells can lose one or more pan-T-cdl antigens, while preserving either a helper/inducer or a cytotoxic/suppressor phenotype. 3 This finding can, in some instances, aid in establishing the diagnosis. We have examined the cell surface antigen phenotype of malignant T cells obtained from the skin, bone marrow, and a lymph node of a 76-year-old man. The site-dependent phenotypic heterogeneity is the subject of this report. REPORT OF A CASE A 76-year-old man had been well until April 1985. when he was found to have numerous plaques and patches involving the 'skin of the trunk and scalp. Results of skin biopsy suggested a diagnosis of lymphoma. The patient was referred to the University of Michigan Hospitals for further evaluation. Physical examination re\'ealed violaceous plaques and poikilodermato us patches, consistent with lymphomatous involvement of the skin. Hepatosplenomegaly was not found. Results of laboratory evaluation were unremarkable, except for the presence of moderate absolute lymphocytosis, with "atypical" forms. Skin, bone marrow, and axillary lymph node biopsies were performed for histologic, flow cytometric, and immunohistochemical analysis.

Received january 6, 1986, from the Department of Pathology. University of Michigan Medical School, Ann Arbor. Revision accepted for publication April 28, 1986. Address correspondence and reprint requests to Dr. Duque: Department of Pathology, College of Medicine, University of Florida, Box j-275, jHMC, Gainesville. FL 32610.

Tissue sections were fixed in neutral buffered formalin and B-5. Paraffin sections were dewaxed and stained with hematoxylin-eosin. For immunohistochemical staining materials fixed in B-5 as well as frozen sections of tissues fixed in acetone were used. T-cell and B-cell markers were assessed in acetone-fixed frozen sections by the avidin-biotin complex (ABC) peroxidase method. 4.5 The reagents, dilutions, and sources of antibodies Til (pan-T), T8 (suppressor/cytotoxic), and T4 (helper/inducer) were used at a I :50 dilution (Coulter Immunology, Hialeah, Florida); K and A immunoglobulin light chains were used at dilutions of I :50 and I: 100, respectively (Becton Dickinson, Mountainview, California). Other cell markers used with frozen sections at a dilution of I: 100 included Leu-I4 (B-cell), Leu-5b, Leu-9 (pan-T-ceIl), and HLA-DR (all from Becton Dickinson). All antibodies were used with the Vector ABC staining kits (Burlingame, California). The procedure used was that described previously for both frozen and paraffin sections,4.5 except that only the paraffin sections were treated with I per cent methanol-hydrogen peroxide for 15 minutes to inhibit endogenous peroxide activity. After washing with phosphate-buffered saline (PBS), sections were treated with 5 per cent normal horse serum to suppress nonspecific binding. Incubation with the antibodies was done for 60 minutes at room temperature. After washing in PBS, the tissues were incubated with the avidin-biotin-peroxidase system, as specified by the manufacturers. For paraffin-embedded tissues, the sections were developed in diaminobenzidine with 0.05 per cent hydrogen peroxide for 10 minutes. For frozen sections, tissues were developed in aminoethylcarbazol (AEC) for 10 minutes. Negative control sections were incubated with purified mouse IgG of the same isotype as the monoclonal antibodies (Miles Laboratory, Kankakee, Illinois). None of the negative controls produced tissue staining. Positive controls consisted of tonsil and spleen for lymphoid markers. Flow cytometric analysis of cell suspensions was performed following gentle teasing of skin, lymph node, and bone marrow. The cell suspensions were subsequently layered over Ficoll/Hypaque to obtain an interface of mononuclear cells. Cell viability, as assessed by trypan blue exclusion, was always more than 95 per cent. Cell surface antigens were demonstrated by indirect immunofluorescence, with fluorescein-conjugated F(ab'h antibody fragments of affinity-purified goat antimouse IgG as the second antiserum. The control consisted of a mouse monoclonal antibody to human glomerular basement membrane (prm'ided by B. Wilson). The cells were analyzed on an EPICS-V flow cytometer equipped with an argon laser at an excitation wavelength of 488 nm; 10,000 cells were examined for each marker. E-rosetle formation was assessed as described previously.6

967

HUMAN PATHOLOGY

Volume 17. No.9 (September 1986)

FIGURE 1 (left). Lymphomatous infiltrate in the dermis consisting of a medium-sized. monomorphic cell type. (Hematoxylin-eosin stain. x 208.) FIGURE 2 (right). Sheep erythrocyte rosette preparation showing resetting around a benign cell and a neoplastic cell. (Wright-Giemsa stain. x 1.100.)

Cell surface marker or flow cytometric analpes were as follows: TIl (thymocyte/mature lymphocyte; T3 (panT, 20 per cent thymoC}'te 8 ; T4 (helper/inducer9 ; T8 (cytotoxidsuppressor8 ; TIO (pan-thymocyte-activated T and B cells8 ; B I (mature B cells9 ; and B4 (B cells lO• All were from Coulter Immunology (Hialeah, Florida). RESULTS Examination of the skin biopsy specimen revealed a monotonous infiltrate consisting of medium-sized lymphoid cells with occasional nuclear angulation and folding (fig. I). The chromatin was dispersed, and nucleoli were inconspicuous. There was no epidermotropism. This lymphoma could not be classified according to the International Working Foundation l1 but had the features of the medium-sized monomorphic lymphoma described by Watanabe et al. 12 Examination of a bone marrow biopsy specimen disclosed a diffuse infiltrate with cytologic features similar to those of the skin lesion. Examination of the lymph nodes showed partial replacement of the paracortical and interfollicular areas with sheets of medium-sized cells that compressed the follicles and germinal centers. Cytologically, the cells were identical to those in the skin and bone marrow. Proliferation of capillary-sized vessels in the paracortical areas was also evident. Plasma cells and eosinophils were rarely seen. Inspection of frozen sections of the skin by the immunoperoxidase method (table I) revealed strong positivity (100 per cent of the cells) for HLA-DR-related antigens

and the T4 phenotype. The pan-T-cell antigens Leu-I, Leu-5, Leu-g, and TIl stained less than 10 per cent of the cells examined. In contrast, immunohistochemical analysis of the lymph nodes revealed that the paracortical areas were intensely positive (approximately 80 per cent of the cells) for Leu-I, Leu-5, Leu-g, and TIl in addition to T4 and HLA-DR. Flow cytometric analysis of cell suspensions (table 2) showed strong positivity for T4 and HLA-DR in the skin, with a T4/T8 ratio of 8.4. Additionally, a high percentage of the cells were T lO-positive. A similar phenotype was observed in the suspension of bone marrow cells, except that TIO was present in only 25% of the cells. In contrast, examination of the cell suspensions obtained from the lymph node revealed two populations, as assessed by forward light scatter (size) and right angle light scatter (internal structure). The phenotype, in both light scatter areas, was that of a mature T cell with T4 positivity. The larger cells, which are presumably the neoplastic component, expressed pan-T-cell antigens to a greater extent. The T4/T8 ratio was 4.4 in the former group and 8.4 in the latter. Fifty-eight per cent of the lymph node cells were Erosette-positive. Furthermore, a cytocentrifuge preparation re\'ealed that both normal and malignant lymphoid cells were E-rosette-positive (fig. 2). DISCUSSION In a stud)' of 50 peripheral T-cell lymphomas, Weiss et a\.3 reported the loss of one or more pan-T-cell antigens in 64 per cent of the cases. This finding has been reported by

968

CASE STUDIES

TABLE 1. Immunohistochemical Analysis of Frozen Tissue

that lea\'e the skin or bone marrow "mature" on entering the microenvironment of the lymph node or, conversely, that the malignant cells of mature phenotype become activated and less mature as they enter the microenvironment of the skin. An alternati\'e explanation for this finding is that the cells that exhibit a mature phenotype in the lymph node are reactive, nonneoplastic lymphocytes. However, se\'eral factors argue against this possibility: 1) The normal T4fT8 ratio in reactive processes is approximately 4: 1. 2 This was, in fact, the ratio that we found in our analysis of the small cells (presumably residual, reactive cells) based on light scatter. In contrast, the larger cells exhibited a T4fT8 ratio of 8.4: 1 (table 2). Thus, in the large cell light scatter gate, the population was enriched for a T4 phenotype, which is that of the malignant cells. The percentage of TIl-positive cells in the large cell area also increased to 63 per cent (table 2); if the large, neoplastic cells were of the same phenotype as those of the skin or bone marrow, a relative depletion of TlI-positi\'e cells should have occurred in this area compared with the small cell area. 2) The E-rosette preparation of a lymph node cell suspension clearly showed a positive reaction in malignant cells. The E-rosette receptor is associated with the TIl antigen,15 and if the malignant cells were TIl-negative, they would not be expected to form E rosettes. This was, in fact, the case in the bone marrow, where TIl expression was seen in 11 per cent of the cells and E rosettes in 18 per cent (table2). Unfortunately, E-rosette formation was not assessed in the cell suspension obtained from the skin. However, with the exception of the degree of TIO positivity, the phenotype in the skin is virtually identical to that in the bone marrow. On the basis of our data, we cannot rule out the possibility that this patient had two malignant clones, each with an incHvidual phenotype. Indeed, biclonality was detected in some B-cell malignancies by Sklar et al. 16 However, with the exception of DNA hybridization techniques to detect rearrangement of T-cell receptor genes!' "c1onality" cannot be detected in T-cell malignancies. Thus, this possibility must be considered. However, if this were the case, tissue localization would be determined by the genotypic or phenotypic characteristics of individual clones or subclones. Our study suggests not only that peripheral T-cell malignancies are heterogeneous with respect to phenotype,

Site ('It positivity) L)mph :-'-odcs

Antibody

Skin

Paracorttx

Ct'nn;lIalern/en

Leukocyte common antigen Leu-I Leu-5 Leu·9 Til T4 TS HLA-DR BI

100 <5 <5 <5 <10 100 <5 100 <5

ND SO 90 60-70 SO 100 <10 100 <10

ND <10 <10 <10 <10 <10 <10 100 100

Abbreviations: ND, not determined,

other investigators. 13 It appears that since there is no specific T-cell phenotypic distribution in peripheral T-cell lymphomas, a wide spectrum of pan-T-cell antigens should be employed. Our case shows the features described by Wood, et aJl3: tumor Ia expression, noncerebriform cytologic features, the absence of epidermotropism, and lack of a mature T-cell phenotype in the skin. With the exception of the lack of epidermotropism, the findings were identical in the bone marrow. \\'e are not aware of previous studies of peripheral Tcell lymphomas in which the phenotype of the lymphoid cells was examined simultaneously in several sites in the same patient. It was of interest that in the lymph node in the present case, but not in the bone marrow or the skin, the malignant cells expressed pan-T-cell antigens. This finding is not completely without precedent, however, inasmuch as Haynes et al. 14 reported a case of cutaneous T-cell lymphoma in which the mature T-cell phenotype was expressed by circulating cells and by the cells infiltrating the skin. However, malignant cells in the skin expressed, in addition, immature T-cell antigens. These immature antigens were not present in the circulating malignant cells. These findings were interpreted as supportive of the notion that the skin can serve as a major site of extrathymic T-cell differentiation. Thus, it can be speculated that immature cells

TABLE 2. Flow Cytometric Analysis of Cell Suspensions Site Lymph :'\otlc

Antibody Leu-I Til T3 T6 TIO T4 TS BI B4 HLA-DR TdT E rosettes T4rrS ratio

(2l)'

Largr Crlls (i9)'

ND 42 46 12 IS 4S II IS 20 4S 0

ND 63 62 3 24 67 S IS 17 51 0

Small Crlls Skin (100)'

12 12 II

I SO 62 15 3 3 94 0 ND S.4

4.4

* ABBREVIATIO:>l: ND, not determined. * Per cent of total cells examined. 969

5S

S.4

Bone

~Iarr()\\"

ND 12 II I ')-

~:>

60 5 2 79 0 0 IS 12

(98)*

HUMAN PATHOLOGY

Volume 17. No.9 (September 1986)

but that within a gi\'en patient the malignant cells can express phenotypic profiles that are site-related. Further studies are needed to determine whether these findings have biologic relevance in terms of revealing homing characteristics and preferential tissue localization of malignant lymphoid cells. REFERENCES I. Waldron]A, Leech]H, Glick AD, et al: ~Ialignant lymphoma of peripheral T-Iymphocyte origin. Immunologic, pathologic and clinical features in six patients. Cancer 40; 1604, 1977 2. Jaffe ES: Pathologic and clinical spectrum of post-th}'mic T-cell malignancies. Cancer In\'est2:413, 1984 3. Weiss L~I, Crabtree GS, Rouse R\', et al: ~Iorphologic and immunologic characterization of 50 peripheral T-ccll lpnphomas. Am ] PathoI1l8:316,1985 4. Taylor CR: An immunohistological study of follicular lymphoma, reticulum cell sarcoma and Hodgkin's disease. Eur] Cancer 12:61, 1976 5. Tubbs RR, Sheibani K: Immunohistology of Iymphoproliferati\'e disorders. Semin Diagn Pathoi 1:272, 1984 6. Kaplan ME, Woodson ~I, Clark C: Detection of human T-lymphoC}'les b}' roseue forming cells. III Bloom B (cd): Ill-vitro ~Iethods in Cell Mediated Immunity. l'\ew York, Academic Press, 1976, PI' 83-88 7. Howard FD, Ledbcuer ]H, Wong], et al: A human T-lymphoC}te differentiation marker defined by monoclonal antibodies that block EroseUe formation.] ImmunoI126:2117, 1981

8. Reinherz EL, Kung PC, Goldstein G. et al: Regulation of the immune response-inducer and suppressor T-IymphoC}te subsets in human beings. l'\ Engl] ~led303:370, 1980 9. Stashenko 1', l'\adler L~I, Hardy R, et al: Characterization of a human B-Iymphocyte specific antigen.] Immunol 125: 1678, 1980 10. l'\adler L~I, Anderson KC, Marti G, et al: B4, a human B-I)"mphoC}'te associated antigen expressed on normal, mitogen-acth'ated, and malignant B-Iymphoc}'tes.] ImmunoI131:24-1, 1983 II. The l'\on-Hodgkin's Lymphoma Pathologic Classification Project: l'\ational Cancer Institute-sponsored study of classification of nonHodgkin's lymphomas: summary and description of a working formulation for clinical usage. Cancer 49:2112, 1982 12. Watanabe S, l'\akajima T, Shimosato Y. et al: T-cell malignancies: subclassification and interrelationships. ]pn] Clin Oncol 9(suppl):423, 1979 13. Wood GS, Burke ]S, Horning S, et al: The immunologic and clinicopathologic heterogeneity of cutaneous lymphomas other than mycosis fungoides. Blood 83:464, 1983 14. Haynes BF, Hensley LL, Jegasothy B\': Differentiation of human TlymphoC}'les I I. Phenotypic difference in skin and blood malignant T-cells in cutaneous T-cell lymphoma.] In\'est Dermalol 78:323, 1982 15. Kamoun M, Martin P], Hanson ]A, et al: Identification of human Tlymphocyte surface protein associated wilh the E-roseue receptor.] Exp Med 153:207, 1981 16. Sklar ], Cleary ~IL, Thiclemans K, et al: Biclonal B-cclilymphoma. l'\ Engl] ~Ied 311:20,1984 17. Bertness \', Kirsch I, Hollis G, et al: T-cell receptor gene rearrangement as clinical markers of human T-ceH lymphomas. l'\ Engl] ~led 313:534, 1985

CORRESPONDENCE Electron Microscopy in Surgical Pathology

To the Editor:-It is unfortunate that in their reply to my letter of June 1985 concerning electron microscopy in surgical pathology Drs. Williams and Uzman chose not to address my "case criticisms." Having dismissed the issue of whether the procedure is as useful as they claim, they instead put forward a justification of the VA electron microscopy program based on other considerations, which should not go unanswered. Their position is that I) electron microscopy is a new tool whose place in practice is not yet established; 2) electron microscopy installations perform many functions, as do the pathologists overseeing them; and, therefore, 3) looking at their previous paper in isolation is invalid and unfairly restrictive. To these assertions I reply that electron microscopy cannot be both so very new that it is to be t~eated financially essentially as research and at the same ~Ime of such proven clinical utility that we should be using It to examine large numbers of specimens at patient expense. Electron microscopy has limited use in surgical pathology, but it is not so poor a thing that it needs special exemption from the critical analysis to which any procedure should be subjected. Drs. Williams and Uzman imply that my questioning, of c~st-effectiveness is superficial because I have failed to conSider all of the other activities centered on the VA e1ect~on ~icroscopes. I am tolerably familiar with such installations m both VA and academic centers and I have no illusions about the demands made on the~. However, if electron microscopy has the limited clinical utility that I infer from the ?riginal article, it is inappropriate to defend its cost-effe,?tlveness because the microscopes are also doing other thmgs at other times. It is never cost-effective to waste time.

There are cross-currents in Drs. Williams' and Uzman's letter confusing research, teaching, and clinical applications of the VA electron microscopy program. The legitimacy of defending part of a program by pleading the existence of other parts may prove unsustainable after critical analysis, Yet is has the potential of transforming an ox to be gored into a sacred cow. L. WOLFSON, MD Hoag Memorial Hospital Presbyterian Newport Beach, California

WILLIAM

MURCS Association

To the Editor:- We read with interest the recent report by Greene et aLI of a patient with the MURCS association and additional congenital anomalies. We recently had the opportunity to examine a 4-day-old infant who died with similar anomalies but who also had central nervous system malformations, which provided further insight into the timing of the embryologic insult. The MURCS association in our patient was manifested at autopsy as uterovaginal agenesis, rudimentary oviducts consisting only of fimbriae, agenesis of the right kidney, left ureteral stenosis with type IV cystic dysplasia of the left kidney, cervicothoracic hemivertebrae and fused vertebrae with a short neck, and rightsided rib abnormalities (absence of ribs I to 3 and fusion of ribs 4 and 5). Also present was a large thoracolumbar meningomyelocele with associated Arnold-Chiari malformation, hydrocephalus, and cervicothoracic syringomyelia. Additional minor anomalies included midline fusion of the adrenal glands, elongated, ribbon-like, histological~y normal ovaries, and preductal coarctation of the aortic arch. Cytogenetic studies showed a normal 46, XX karyotype.

970