British journal of Huematofogy. 1994, 86, 659-662

SHORT REPORT

Aggressive natural killer cell leukaemia/lymphoma in two patients with lethal midline granuloma

J E S U S SOLER, RAMON BORDES,’ FRANCISCO ORTONO,* MARIO MONTAGUD,$ J A U M E MARTORELL, CRISTINA PONS,’ JOSEP NOMDEDEU, J U A N JOSE LOPEZ-LOPEZ,~ J A I M E PRAT’ A N D MIOUEL RUTLLANT Servei d’tiematologia, ’Servei d’Anatomia Patologica, ’Servei d’Oncologia, Hospital de la Santa Creu i Sant Pau. Barcelona, and 3Servei d’lmmunologia, Hospital Clinic Provincial, Barcelona, Spain

Received 6 August 1993; accepted for publication 18 November 1993

Summary. We report two patients with leukaemic prolifera- tions of large granular lymphocytes. The immunophenotype study showed that the leukaemic cells were positive for CD2. CD38. CD56 and anti-HLA-DK monoclonal antibodies and negative for other T-cell (CD3. CD4, CD8) and B-cell markers (CDI 9, CD20 and surface immunoglobulins). The clinical course was acute and a diagnosis of aggressive natural killer cell leukaemia/lymphoma was made. No clonal rearrange- ments ofeither C g T-cell receptor or J,, immunoglobulin genes

were found. Functional studies done in one patient demon- strated non-restricted cytotoxic activity after activation with IL-2. Lethal midline granuloma had been previously diag- nosed in both patients. A possible relationship between this entity and the natural killer cell leukaemia is discussed.

Keywords: large granular lymphocytes, natural killer leukae- mia, lethal midline granuloma, angiocentric lymphomas.

Aggressive natural killer cell leukaemia/lymphoma (ANKL/ L) is a new clinicopathological entity which represents a rare type of large granular lymphocyte leukaemia derived from a natural killer (NK) cell subset (Imamura et al. 1990). Patients present with fever, hepatosplenomegaly and lymphadeno- pathy. Anaemia, neutropenia and thrombocytopenia are common. Increased numbers of lymphoid cells with basophi- lic cytoplasms containing numerous azurophilic granules (LGI, cells) appear at the onset or during the course of the disease. These cells are positive for CD2, CD38. C D 5 6 and anti-HLA-DK monoclonal antibodies and negative for T-cell and B-cell markers. Functional studies demonstrate that the cells have NK activity and immunogenotypic studies show no rearrangements of TB, T6. TT or immunoglobulin genes (Imamura et a/, 1990). We describe two cases of ANKL/L which developed in patients in whom lethal midline granu- loma (LMG) had been previously diagnosed. This entity embraces an uncommon and heterogenous group of dis- orders characterized by a progressive and relentless destruc-

* Present address: Servico de Hematologia. Hospital General.

$ Present address: Servico de Hematologia, Hospital Clinico. Centro Regional de Hemodonacion. Murcia. Spain.

Valencia. Spain.

Correspondence: Dr J. Soler. Servei d’Hematologia. Hospital de la Santa Creu i Sant Pau. Avgda S.A.M. Claret 167, 08025 Barcelona, Spain.

tive ulceration and by a necrosis limited to the midline upper respiratory structures. LMG has been included in the group of angiocentric immunoproliferative lesions (AIL) which have been considered to be a distinct group of T-cell lymphomas (Jaffe, 1984: Ishii et a!, 1982; Lipford et ul, 1988). We investigate the possible connection between LMG and ANKL/L.

CASE REPORTS Case 1. A 64-year-old woman was admitted to our hospital

in November 1984 with a l0-month history ofnasal bleeding which was unresponsive to symptomatic treatment. The patient was otherwise well. Physical examination was unremarkable as was her medical history. Laboratory data were as follows: Hb 15.0 g/dl: WBC 8.0 x 10y/l with 0.74 neutrophils, 0.18 lymphocytes and 0.08 monocytes. The platelet count was 307 x 10y/l. Her kidney and liver function were normal. The immunoglobulin values were within the normal range. A rhinoscopy was performed and a biopsy of the upper respiratory airways mucosa was taken. Non- necrotizing focal ulceration and a heavy infiltrate consisting of atypical lymphoid cells, some plasmatic cells and eosinophils were observed. No angiocentric distribution of the infiltrate was noted. A culture of the mucosal biopsy samples was negative. A diagnosis of polymorphic reticulosis was made. Bone marrow biopsy was normal. The patient received local

659

660 Short Report radiotherapy (50 Gy) achieving complete remission. Five months later, a subcutaneous node was noted in the right leg. A biopsy showed a heavy infiltrate due to atypical lymphoid cells similar to those observed in the first nasal biopsy but with a clear angiocentric distribution. The rest of the physical examination and the laboratory data were unremarkable. A bone marrow biopsy was normal. Local radiotherapy (50 Gy) and six cycles of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) chemotherapy were administered, achieving a second remission. In November 1986, multiple subcutaneous nodules were noted in both legs. Cyclophos- phamide, vincristine and prednisone (CVP) chemotherapy was administered without response. Five months later, progressive liver enlargement was noted and multiple enlarged cervical, axilar and inguinal lymph nodes were palpated. In July 1987, a peripheral blood count showed WBC 51 x 10y/l with 0.88 LGL cells. A bone marrow biopsy showed a diffuse infiltrate of these cells. The patient died 1 month later. Autopsy disclosed polypoid enlargements of the basal and maxillary sinus mucosa. Microscopic examination of both mucosas revealed an infiltrate of atypical lymphoid cells with an angiocentric distribution, some plasma cells and eosinophils. An atypical infiltrate with the same histologic and cytologic characteristics was also observed in the skin, liver, spleen, lymph nodes and bone marrow.

Case 2 . A 45-year-old man was admitted to our hospital because of a 2-month history of bloody rinorrhoea, right ptosis and diplopia. Physical exploration disclosed a marked proptosis of the right eye. Standard blood and biochemical studies were normal, including the lactic-dehydrogenase level. He was HIV negative. Computed tomography (CT) revealed local invasion of the right etmoidal sinus and the right orbit. Chest radiography and abdominal CT were normal, The nasal mass was biopsed and the histopathologi- cal study revealed a diffuse infiltrate of atypical lymphoid cells suggestive of malignant lymphoma with an angiocentric pattern. Bone marrow biopsy was normal. The patient received standard first-line chemotherapy (CHOP) associated with treatment of the central nervous system (methotrexate 1 5 mg), achieving a partial and brief response. A month later the sinusal mass increased markedly and third-line chemotherapy-prednisone. methotrexate, leucovorin, doxorubicin, cyclofosfamide, etoposide (PROMACE)-was administered together with local radiotherapy (SO Gy). At the end of the second cycle of chemotherapy he developed thrombocytopenia and lymphocytosis. The blood findings were: haemoglobin: 12 .4 g/dl; WBC 8 . 0 ~ 109/1 with 0.02 band forms, 0.07 neutrophils. 0.80 of atypical lympho- cytes, 0 .04 monocytes. 0.04 metamyelocytes, 0 . 0 3 myelo- cytes; 11 erythroblasts/100 WBC: platelets 49 x 10q/l. The general condition of the patient deteriorated rapidly. He developed acute liver failure and died 3 months after the diagnosis. Results of a biopsy performed in the last week of his fatal disease revealed hepatic and bone marrow infiltra- tion by the same process.

MATERIALS AND METHODS Immunohistochemical studies were carried out using a n immunoperoxidase linked avidin-biotin-complex (Vector)

method. The following antibodies were studied: CD3 T-cell polyclonal (CD3) Dako. Leu M1 (CDlS) Becton Dickinson; L2h (CD20) Dako, 2H4 (CD45RA) Coulter Clone, UCHLl (CD45RO) Dako; LN2 (CD74) Bio-test and Mac 387, Dako. In case 1 immunohistochemical studies were carried out on paraffin sections of the nasal mucosa at diagnosis, the first subcutaneous biopsy and on sections of infiltrated bone marrow, whereas in case 2 they were done only in the initial nasal biopsy. Surface membrane markers of peripheral blood leukaemic lymphocytes were studied using direct and in- direct immunofluorescence, and examined with the aid of a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.). The monoclonal antibodies studied are shown in Table I. Electron microscopy (EM) studies were carried out using the paraffin blocks of the nasal biopsy. Immunogenotypic studies were performed according to the Southern method using a C/I probe for the T-cell receptor genes (Dr T. W. Mak. Ontario) and a J H probe for the immunoglobulin genes (Dr T. H. Rabbitts, Cambridge). DNA was prepared using standard procedures. In case 2, surface membrane markers and immunogenotypic determinations were carried out with cryopreserved mononuclear cells taken during the leukaemic phase (July 1987).

RESULTS

In both cases immunohistochemical studies revealed that atypical lymphoid cells were positive for the IJCHLI (CD45RO) and for LN2 (CD74) antibodies and negative for the other antibodies (CD3, CD15. CD20, CD45RA) and Mac 387. Results of surface membrane markers are shown in Table I. The EM of the tumour cells showed several fixation artefacts. Granules resembling lysosomes were detected but tubulo-reticular structures were not observed. In case 2 , functional studies demonstrated no detectable spontaneous NK cytotoxic activity against K 562 cell line. However, non- restricted cytotoxic activity was observed after activation with IL-2. No clonal rearrangements using the C,4 probe were found in either case.

DISCUSSION

ANKL/L usually occurs as a de n o w leukaemia, although cases with a very closed clinical and biological picture have been described as a n acute transformation of chronic large granular cell leukaemia (Ohno et ul, 1989) and in the terminal stage of cutaneous lymphomas (Seno et al. 1992). We report two cases in which the ANKL/L developed in patients with a previous diagnosis of LMG. Both patients presented with a n angiocentric pattern characteristic of LMG, although in patient 1 this pattern was not seen at diagnosis probably because of the poor quality of the sample.

Given the clinical. morphological and immunophenotypi- cal features of our patients, we feel that they suffered from a unique lymphoproliferative disorder which initially pre- sented as LMG before becoming leukaemic. This is evident in case 2 because of the short interval between LMG and the leukaemic phase. However, in case 1 this interval exceeded 2 years, with the result that the relationship between LMG and

Short Report 661 Table I. Results of the immunophenotypic studies in the peripheral blood LGL cells.

Antibody Source

Results

Cluster Case 1 Case 2

Leu6 T11 T3 T4 Crisl Leu9 T8 LFA-1 a Leu1 5 LeuM 5 LeuM 1 Leu1 1 LFA-1B B1

134-2C2 4B4 IOB6 33-3B3 2H4 UCHLl Leu19 Leu 7 anti6 Leu8 Edu 1 WT-3 1 TdT

IL-2R

Becton Dickinson Coulter Clone Coulter Clone Coulter Clone Dr R. Vilella Becton Dickinson Coulter Clone Janssen Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Janssen Coulter Clone Becton Dickinson Dr R. Vilella Coulter Clone Immunotech Dr R. Vilella Coulter Clone Dako Becton Dickinson Becton Dickinson T-cell Sciences Becton Dickinson Dr R. Vilella Becton Dickinson Supertechs

CD1 CD2 CD3 CD4 CD 5 CD7 CD8 CDlla CDllb CDl1 c CD15 CD16 CD18 CD20 CD2 5 CD2 6 CD29 CD38 CD44 CD45RA CD45RO CD56 CD57 -

+

ND ND

+ - - ND ND + - - ND + + + ND +

-

- ND +

+

+ : more than 50% cells positive: -: less than 10% cells positive. Edul : anti- HLA-DR. WT-31: anti-fi chains of T-cell receptor.

the ANKL is not so clear. In these cases the morphological studies suggest that the neoplastic cells were the same throughout the evolution of the disease. The atypical lym- phoid cells that appeared in the skin, lymph node and bone marrow resembled, from the histological view point, those cells observed in the upper respiratory tract at diagnosis. Nevertheless, since it is not possible to determine the LGL morphology in routine histology, the morphological similar- ity between LMG and leukaemic LGL cannot be proved beyond reasonable doubt. We carried out ultrastructural studies using the paraffined material of nasal biopsies which disclosed numerous lysosomes in the cytoplasm of the lymphoid cells. This might point to the granular nature of the lymphoid cells in the LMG.

It is not possible to compare fully the immunophenotype of atypical lymphoid cells of LMG lesions with that of leukaemic cells. since most of the monoclonal antibodies used in cell suspensions do not work on paraffin sections. Immunohisto- chemical studies using paraffin sections of nasal biopsies disclosed that atypical cells were positive for 0 7 4 and CD45RO and negative for CD3, CDI 5, CD20, CD45RA and Mac 387. This phenotype is not suggestive of T-cell, B-cell or histiocytic lineage but it is fairly similar to that observed in

leukaemic cells (Table I) and to that reported for ANKL/L (Imamura et ul, 1990). Thus, the immunohistochemistry suggests that the atypical lymphoid cells in the LMG lesions and peripheral blood were of the same origin.

We conclude that in our two cases the ANKL/L may represent the terminal event in the evolution of LMG. This is not surprising, since immunological characterization of AIL disorders affecting upper respiratory tracts has demonstrated an immunophenotype similar to that of NK cells in most cases studied (Ng et a/. 198 7,1988; Ho et al, 1990: Kanavaros et aI, 1993). Although the aetiology of LMG is unknown, recent evidence suggests that EBV may play a role in the develop- ment of LMG, which points to the possibility that these patients may have an occult immunodeficiency (Borisch et al, 1993: Peiper, 1993: Kanavaros et al, 1993).

ACKNOWLEDGMENTS

We are grateful to Drs R. Vilella, Hospital Clinic Provincial, Barcelona, for providing monoclonal antibodies and to Dr T. W. Mak and T. H. Rabbitts for providing the DNA probes.

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