Dr Estella Matutes, Academic Department of Haematology and Cytogenetics, The Royal Marsden NHS Trust, Fulham Road, London SW3 6JJ, UK. E-mail: email@example.com
We describe a case of T-cell large granular lymphocyte (LGL) leukaemia that transformed into a large-cell T-cell lymphoma 11 years from diagnosis. A 29-year-old asymptomatic female presented in 1989 with lymphocytosis, neutropenia and mild bone marrow infiltration. The circulating cells were LGL with a CD2+, CD3+, CD8+, CD4–, CD16+, CD56+, CD57– phenotype. In August 2000, she developed fever, a large submandibular mass and hepatosplenomegaly. Biochemistry showed abnormal liver function tests and raised lactate dehydrogenase (LDH) levels. A serological screen for Epstein–Barr virus, cytomegalovirus, human T-lymphotropic virus-I, human herpes virus (HHV)-6 and HHV-7 was negative. Histology of the mass was consistent with the diagnosis of peripheral T-cell lymphoma composed of large cells, and immunohistochemistry showed that the lymphoma cells had a phenotype identical to the mature LGL. Molecular analysis with the polymerase chain reaction (PCR) demonstrated rearrangement of the T-cell receptor (TCR) γ-chain gene with a band of identical size in both bone marrow mature LGL and lymph node cells. The patient was treated with CHOP (cyclophosphamide, vincristine, doxorubicin and prednisolone), resulting in the disappearance of the mass and improvement of the hepatosplenomegaly, LDH and liver abnormalities. She underwent splenectomy, and spleen histology showed involvement by T-cell LGL leukaemia with no evidence of transformation. This case illustrates that transformation or Richter syndrome may occur in a minority of patients with T-cell LGL leukaemia, a disease that has a benign clinical course in most cases. This is the first case documented by molecular methods of the transformation of the pre-existing clone.
Large granular lymphocyte (LGL) leukaemia is a neoplastic disorder that results from the clonal expansion of cells with distinct morphology, i.e. LGL. There is certain immunophenotypic heterogeneity in this disease. The majority of cases express functional T-cell antigens, rearrange the T-cell receptor (TCR) chain genes and have a mature cytotoxic T-cell phenotype (CD2+, CD3+, CD8+, CD57+, CD16+/–). A minority of LGL have a natural killer (NK) immunophenotype (CD2+, CD3–, CD7+/–, CD56+) without evidence of TCR rearrangement (NK– LGL) (Loughran, 1993). The latter form is prevalent in the eastern contries, tends to have a more aggressive course and, in some cases, has been shown to be associated with Epstein–Barr virus (EBV).
T-cell LGL leukaemia runs, as a rule, a protracted or chronic clinical course without the need for therapy in asymptomatic patients. Immunosuppressive agents, e.g. cyclosporine A, methotrexate or cyclophosphamide, are the treatments used when symptomatic cytopenias or progression do occur. The median survival is estimated to be > 10 years in various series (Dhopdapkar et al, 1994; Lamy & Loughran, 1998). Cases of LGL leukaemia with aggressive course have been documented, but the majority of these had the NK– LGL form (Ohno et al, 1988, 1989; Imamura et al, 1990; Hart et al, 1992; Toba et al, 1997), and only a few presented with a T-cell LGL lymphoma (Brito-Babapulle et al, 1987; Macon et al, 1996) or a CD3+, CD56+ aggressive T-cell variant of LGL leukaemia (Gentile et al, 1994). All these patients manifested with aggressive disease without evidence of a preceding asymptomatic long phase that characterizes T-cell LGL leukaemia. The term ‘NK-like’ LGL lymphoma or leukaemia has been proposed to designate the subgroup with aggressive course (Macon et al, 1996).
Transformation of T-cell LGL leukaemia into a high-grade T-cell lymphoma has not been well recognized. In the few cases in which there was a dual population of blast cells and mature LGL (Brito-Babapulle et al, 1987) and/or a morphological change from LGL to blast-looking cells (Tagawa et al, 1992), there was not sufficient evidence by molecular or cytogenetic analysis to show that both cells derived from the same clone and, in one case, the cell lineage was ambiguous (Nowell et al, 1981).
We describe a patient with a long-standing T-cell LGL leukaemia who, 11 years after diagnosis, developed a peripheral T-cell lymphoma of large-cell type. Immunophenotyping and molecular genetics demonstrated the identical origin of the mature LGL and the large lymphoma cells.
A 29-year-old female was found to have lymphocytosis and neutropenia in 1989. Physical examination did not show organomegaly. The white blood cell (WBC) count was 8 × 109/l, lymphocytes 5 × 109/l and neutrophils 1·4 × 109/l; haemoglobin (Hb), platelet count, erythrocyte sedimentation rate (ESR), liver and renal biochemistry were normal. A bone marrow aspirate showed infiltration by LGL cells with similar morphology and immunophenotype to the cells in the blood, and the bone marrow trephine biopsy showed an interstitial lymphoid infiltrate with the presence of normal numbers of erythroid precursors, megakaryocytes and reduced myelopoiesis. The patient remained asymptomatic with no therapy except for a susceptibility to infections linked to the neutropenic periods, which reached 0·2 × 109/l on some occasions. In the interim, she had four pregnancies with transient improvement of the neutrophil counts on each occasion. Eleven years after presentation, she developed intermittent fevers, B symptoms and a large submandibular mass. Physical examination showed splenomegaly, 20 cm below the costal margin, hepatomegaly and submandibular lymphadenopathy measuring 5 × 8 cm. Peripheral blood counts were: Hb 9·7 g/dl; platelets 130 × 109/l and WBC 1·6 × 109/l with 65% neutrophils and 30% lymphocytes. Biochemistry showed raised alkaline phosphatase (722 IU/l), gamma glutamyl transpeptidase (427 IU/l), bilirubin (25 µmol/l), ESR (60 mm), lactate dehydrogenase (LDH, 2001 IU/l) and Beta2 microglobulin (3·1 mg/l). Renal function tests including serum calcium levels and serum immunoglobulins were normal. Serology for hepatitis B and C, EBV (IgG and IgA), human T-lymphotropic virus (HTLV)-I, HTLV-II, human herpes virus (HHV)-6 and HHV-7 was negative. There was evidence of a past infection by cytomegalovirus (CMV) with positive IgG but negative CMV early antigen in the buffy coat. Autoimmune screen showed a weakly positive rheumatoid factor with a titre of 25 IU/ml. Coomb's test was negative. Chest X-ray was normal, and a computerized tomographic (CT) scan confirmed spleen and liver enlargement without focal abnormalities and no evidence of chest, abdominal, retroperitoneal and pelvic lymphadenopathy. A bone marrow aspirate, trephine biopsy and lymph node biopsy were performed, and the patient was treated with CHOP (cyclophosphamide, vincristine, doxorubicin and prednisolone) with resolution of the neck mass, improvement of the liver function tests and LDH but persistence of the hepatosplenomegaly. During the first two courses, the patient had two severe episodes of sepsis with fever, hypotension and disseminated intravascular coagulation (DIC) that required intensive care. She recovered with supportive care, broad-spectrum antibiotics and antifungals. No microorganisms were isolated in blood, urine and/or stool samples. The patient underwent splenectomy with no complications and significant improvement of the peripheral blood counts and, later, a liver biopsy was performed. Seven months from transformation, she is alive, well with no B symptoms, although she still has moderately abnormal liver function tests and hepatomegaly.
Materials and methods
Morphology This was examined on May–Grunwald–Giemsa-stained peripheral blood and bone marrow films at diagnosis and at transformation. Histology of the bone marrow trephine and lymph node was analysed in haematoxylin–eosin sections.
Immunological markers These were performed by flow cytometry in isolated peripheral blood and bone marrow mononuclear cells at diagnosis and at the time of transformation. Immunohistochemistry was carried out on routinely formalin-fixed, paraffin-embedded material from the bone marrow trephine, lymph node biopsy and spleen using a diaminobenzidine (DAB) modified biotinylated/avidin method with a basic kit from the manufacturers on the Ventana automated immunostainer. Heat-mediated antigen retrieval and protease enzyme digestion were used to unmask antigens where appropriate. The following monoclonal antibodies (mAbs) were used: CD2, CD3, CD5, CD7, CD4, CD8, CD11b, CD16, CD56, CD57, CD38, CD25, HLA-Dr, anti-TCR and CD19 to test blood and bone marrow cells in suspension; and CD3, CD5, CD4, CD8, CD20, CD30, CD56, CD57, TIA-1 and EBV LMP-1 in the tissue sections.
Molecular analysis This carried out in bone marrow and lymph node tissue sections by a polymerase chain reaction (PCR) using primers for the TCR gamma-chain genes. DNA was extracted from paraffin-embedded samples of lymph node and bone marrow as described previously (Diss et al, 1994). T-cell clonality analysis was performed using two sets of primers directed to the variable and joining regions of the T-cell receptor gamma-chain gene (Diss et al, 1995). Set 1 contained primers VG-I, VG-II, VGIII/IV and JG-12, and set 2 contained primers VG-I, VG-II, VGIII/IV and JPG-12 as shown in Table I (Goudie et al, 1990; McCarthy et al, 1992).
Table I. Primer details.
TCT GG (G/A) GTC TAT TAC TGT GC
GAG AAA CAG GAC ATA GCT AC
CTC ACA CTC (C/T) CA CTT C
CAA GTG TTG TTC CAC TGC C
GTT ACT ATG AGC (T/C) TA GTC C
Test samples were analysed in parallel with a T-cell lymphoma-positive control and a negative control without template DNA and run on 10% polyacrylamide gels, stained with ethidium bromide and viewed under ultraviolet light.
The majority of lymphocytes in the blood and bone marrow aspirate at diagnosis and at the time of transformation were LGL with eccentric nucleus, mature nuclear chromatin and abundant cytoplasm with azurophilic granulation (Fig 1). There were no circulating large cells.
At diagnosis and at transformation, histology of the bone marrow showed an interstitial infiltrate composed of small mature-looking lymphocytes; large cells were not present. Lymph node histology showed diffuse replacement of the normal architecture by pleomorphic large cells with immature chromatin, irregular nucleus and prominent nucleoli. Mitoses were easily identified (Fig 2.) Spleen histology showed abundant extramedullary haemopoiesis. Additionally, there was an infiltrate within the red pulp and in the sinusoids consisting of small to intermediate-sized cells with scattered occasional larger cells that were not present in clusters. The liver biopsy showed preservation of the architecture with dilated sinusoids and scattered small lymphocytes.
In the peripheral blood and bone marrow, these showed a mature T-cell phenotype. A proportion of cells > 70% were CD2+, CD3+, CD8+, anti-TCR+, CD38+ and CD56+; half of these cells were also CD11b+, CD16+, and a third of them were CD5, CD7 and HLA-Dr positive; cells were negative with CD4, CD57, CD19 and CD25 (Table II). In conclusion, the phenotype was that of a mature cytotoxic T cell (CD3+, TCR+, CD8+, CD16+, CD56+).
Table II. Immunological markers (peripheral blood and bone marrow).
Positive cells (%)
Immunohistochemistry in the lymph node tissue sections showed that the large cells were positive for CD3, CD5, CD8, CD56 and CD30 and expressed the cytotoxic granule-associated protein TIA-1 (Fig 3); CD4, CD57 and LMP-1 were negative, and CD20 stained small aggregates of residual B cells. Cells in the bone marrow trephine biopsy had a CD3+, CD8+, CD56+, CD4–, CD57–, CD30– phenotype identical to the circulating mature LGL (Fig 4). In the spleen, the small cells expressed CD3, CD5, CD8 and CD56, whereas the occasional larger cell expressed these markers with the addition of CD30
On the bone marrow and lymph node, molecular analysis showed a rearrangement of the TCR γ-chain gene with the presence of a band of identical size in the two tissues examined (Fig 5).
We describe an unusual case of T-cell LGL leukaemia which, 11 years after diagnosis, transformed into a large-cell peripheral T-cell lymphoma. Transformation of and/or aggressive course in LGL leukaemia has been documented (Ohno et al, 1988, 1989; Imamura et al, 1990; Hart et al, 1992; Toba et al, 1997), but all these cases had an NK phenotype with germline configuration of the TCR chain genes. In such patients, transformation occurred at 5 months to 6 years from presentation, and prognosis was poor. In contrast, transformation in the most common form of T-cell LGL leukaemia has not been well recognized. Nowell et al (1981) described a patient with clonal lymphocytosis who, at 5 years, developed an immunoblastic lymphoma. Although clonal evolution was demonstrated by cytogenetics, the lineage of the cells was uncertain. The lymphocytes at presentation were CD2+ (E-rosette+), whereas the lymphoma cells had a ‘null’ phenotype (CD2–). Therefore, it cannot be ruled out that this case corresponded to the NK– LGL form. Tagawa et al (1992) documented a case with a T-cell phenotype who, 22 months after diagnosis, transformed with circulating blast cells; however, no clonal evolution was documented, and cytogenetics showed a different karyotype in the cells at the two phases of the disease course. Matsubara et al (1994) reported a case of LGL leukaemia with an aggressive evolution at 7 months, but evidence of transformation was equivocal by histology, as this showed liver infiltration by mature LGL, and molecular/cytogenetic analysis was not carried out. We have previously documented a case of CD8+ LGL leukaemia/lymphoma with aggressive course and a mixture of circulating mature LGL and immunoblasts. Although the phenotype in this case was consistent with a T-cell lineage origin of the cells, TCR analysis failed to demonstrate rearrangement of the β/γ-chain genes, and cytogenetic studies, showing a complex karyotype, were performed in specimens containing a mixture of mature LGL and blasts. Therefore, we could not conclude that the same abnormality was present in both mature and immature cells (Brito-Babapulle et al, 1987). In contrast, the patient described here had a long-standing asymptomatic T-cell LGL leukaemia that transformed into a large T-cell lymphoma as demonstrated by histology, immunophenotype and molecular analysis of the TCR chain genes. Thus, lymph node histology showed that the majority of cells were large cells with blastic morphology and had a similar phenotype to the LGL cells except for the CD30 expression, the latter probably reflecting cell activation. In addition, molecular analysis showed T-cell clonality with the presence of a band of identical size in the LGL and blasts. Our case has some similarities to but also differences from the cases described as aggressive variants of T-cell LGL leukaemia with a CD3+ CD56+ phenotype (Gentile et al, 1994). Similar to the T-cell LGL variant, the main manifestations at transformation in our patient were fever, splenomegaly, lymphadenopathy, a T-cell phenotype (CD3+, CD8+, CD57–, CD56+) and rearrangement of the TCR; however, in the aggressive LGL variant, patients had high WBC counts with no evidence of blast-looking cells in the blood or tissues examined and had a rapidly progressive course from presentation (Gentile et al, 1994).
T-cell LGL leukaemia is a chronic indolent disease in the majority of patients. Our case illustrates that the disease may transform rarely, a phenomenon similar to Richter transformation in CLL. The secondary events that led to transformation in our patient are so far unknown. EBV has been implicated in some cases of aggressive NK– T-cell leukaemias (Hart et al, 1992; Toba et al, 1997) and in Richter transformation of CLL (Ansell et al, 1999). However, negative EBV serology and lack of LMP-1 protein expression in the large cells do not support the involvement of this virus. In one case of T-cell LGL, the aggressive course was associated with marked reactivation of the herpes virus HHV-6 (Tagawa et al, 1992), but serology for this virus in our patient was negative. LGL has very rarely been associated with HTLV retroviruses (Loughran et al, 1994), but serology was negative in our patient.
In summary, we report a case of T-cell LGL leukaemia in which transformation into a large-cell lymphoma was well documented by histology, immunophenotype and molecular analysis.