Hepatosplenic γδ T-cell lymphoma after liver transplantation: Report of the first 2 cases and review of the literature

Abstract

Hepatosplenic γδ T-cell lymphoma is a rare lymphoproliferative disorder originating from natural killer–like Vδ1-lymphocytes. This subtype has been described after different types of solid organ transplants. In this article, we describe the first 2 cases after liver transplantation. Both patients had thrombocytopenia with (hepato)splenomegaly but without peripheral lymphadenopathies and sinusoidal infiltration of the liver and spleen by monomorphic γδ-lymphocytes on pathological examination. The clinical and pathological findings, immunophenotypical profile, prognosis, and treatment are highlighted. In order to make an early diagnosis, physicians who take care of liver transplant recipients should be aware of the characteristic features of this posttransplant lymphoproliferative disorder. Therefore, a diagnostic algorithm is proposed. Liver Transpl 15:686–692, 2009. © 2009 AASLD.

Patients receiving long-term immunosuppressive therapy after solid organ transplantation are at risk for developing posttransplant lymphoproliferative disorders (PTLDs). The majority of these proliferations are of B-cell origin and are associated with Epstein-Barr virus (EBV) infection. T-cell PTLDs are comparatively rare and account for less than 15% of all lymphomas after transplantation. In contrast to B-cell PTLDs, they are less frequently associated with EBV and are more often found extranodally.1

According to the World Health Organization Classification of Tumours (2001), hepatosplenic T-cell lymphoma is recognized as a distinct clinicopathological entity among the peripheral T-cell lymphomas on the basis of the expression of T-cell receptors (TCRs; γδ-TCR and minority αβ-TCR), the clinical presentation, and the pattern of histological involvement.2 Subsequently, a number of cases of hepatosplenic γδ T-cell lymphoma (HSγδTCL) have been described, though mainly as case reports and in small series. Recently, 2 large series have been published. In these series, a small proportion of cases of HSγδTCL after solid organ transplantation, especially kidney transplantation, were described.3, 4 However, to the best of our knowledge, no case has been described following liver transplantation. We describe here the first 2 cases of post–liver transplantation HSγδTCL and discuss the features that may help transplant physicians with the early recognition of this disorder.

CASE 1

A 23-year-old female patient was hospitalized with fever and decline of her clinical condition. Her medical history revealed a liver transplant 6 year before admission due to fulminant idiopathic hepatitis (EBV-negative). Two years later, she developed severe cellular rejection while receiving cyclosporine and azathioprine; her therapy was switched from cyclosporine to tacrolimus. Six months later, an association of mofetil mycophenolate instead of azathioprine was deemed necessary because of persistent rejection. Mofetil mycophenolate was subsequently replaced by azathioprine again because of drug tolerability problems.

On admission, the patient had a fever (38.8°C), watery diarrhea, cough with purulent sputum, and general myalgia. Clinical examination revealed sinus tachycardia (118 beats per minute) with normal arterial tension (100/70 mm Hg). Her abdomen was diffusely bloated with significant hepatosplenomegaly. Subcutaneous bleeding and ecchymosis on both arms were noticed.

Laboratory results showed normochromic, slightly macrocytic anemia, thrombocytopenia, and a normal leukocyte count. Differentiation showed normoblasts (11%) with clear anisocytosis. Other abnormalities included hypoalbuminemia and elevated alkaline phosphatase, C-reactive protein, and lactate dehydrogenase (Table 1, patient 1). Immunophenotyping of peripheral blood showed a markedly elevated percentage of T-lymphocytes. The patient was EBV-seropositive. Salmonella group D was detected in her stool.

Table 1. Blood Results

	Patient 1	Patient 2	Reference
Abbreviations: γ-GT, gamma glutamyl transpeptidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; LDH, lactate dehydrogenase; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.
Hemoglobin (g/dL)	8.0	6.9	12.0–16.0
MCV (fL)	99.6	83.7	76–96
MCH (pg)	31.1	27.7	27–32
Thrombocytes (10^9/L)	76	485	150–450
White blood cells (10^9/L)	8.8	4.1	4.0–10.0
LDH (U/L)	2040	968	240–480
CRP (mg/L)	229	180	<5
Alkaline phosphatase (U/L)	385	694	<240
γ-GT (U/L)	40	176	<35
AST (U/L)	86	74	<32
ALT (U/L)	12	125	<31
Total bilirubin (mg/dL)	0.74	4.0	<1.0
Direct bilirubin (mg/dL)	—	2.8	<0.5
Albumin (g/L)	21.1	37.6	35–52
Creatinine (mg/dL)	0.84	1.83	0.51–0.95
Uric acid (mg/dL)	2.7	10.4	2.6–6.2

Hepatosplenomegaly with multiple para-aortic, peritoneal, and mesenterial adenopathies was visualized by computed tomography (CT), whereas fluorodeoxyglucose positron emission tomography revealed hot spots at the liver, spleen, and bone marrow (Fig. 1A). Flow cytometric analysis of a bone marrow smear demonstrated increased cellularity with an invasion of blastlike cells, with 41% TCRγδ+, CD2+, CD3+, CD4−, CD5−, CD7+, CD8−, and CD56+. The diagnosis of HSγδTCL was confirmed by biopsy of the liver, which revealed sinusoidal dilatation infiltrated by monoclonal γδ T-lymphocytes, while the portal tracts were spared (Fig. 2). Further investigation of the liver biopsy specimen showed clonal rearrangements of TCR-β, TCR-γ, and TCR-δ genes (detected by polymerase chain reaction and confirmed by Southern blotting), isochromosome 7q, and trisomy 8.

Figure 1. Positron emission tomography scintigraphy: (A) hot spots at the liver, enlarged spleen, and pelvic area at the diagnosis of hepatosplenic T-cell lymphoma and (B) no more disease activity after allogeneic stem cell transplantation.

Figure 2. Liver biopsy shows portal tract vein (V), artery (A) and bile duct (BD) without signs of acute or chronic rejection. There is a marked infiltration by monomorphic lymphocytes in the dilated sinusoids (arrows) with presence of 1 mitotic figure (arrowhead 40×).

Treatment with ProMACE (prednisone, methotrexate, doxorubicin, cyclophosphamide, and etoposide) and CytaBOM (cytarabine, bleomycin, vincristine, and methotrexate) was initiated. However, because of disease progression after 2 cycles, treatment was switched to fludarabine, mitoxantrone, and dexamethasone. Because of tumoral progression, therapy with alemtuzumab (anti-CD52 antibodies) was attempted, followed by 2 cycles of DHAP (cisplatinum, cytarabin, and dexamethasone).

Six months after diagnosis, the patient received an allogeneic human leukocyte antigen–matched stem cell transplant from a human leukocyte antigen–identical sibling, which resulted in complete remission (Fig. 1B). The posttransplantation period was complicated by grade II graft-versus-host disease, Clostridium difficile–associated pseudomembraneous colitis, herpes zoster infection, and streptococcal bacteremia. Despite increasing doses of immunosuppressive therapy, chronic graft-versus-host disease persisted. Twelve months after the diagnosis of HSγδTCL, the patient died because of invasive pulmonary aspergillosis.

γ-GT, gamma glutamyl transpeptidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATG, antithymocyte globulin; CHOP, cyclophosphamide, doxorubicin, vincristine, and methylprednisone; CMV, cytomegalovirus; CRP, C-reactive protein; CT, computed tomography; EBV, Epstein-Barr virus; HSγδTCL, hepatosplenic γδ T-cell lymphoma; LDH, lactate dehydrogenase; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; PET, positron emission tomography; PTLD, posttransplant lymphoproliferative disorder; TCR, T-cell receptor; TMA, thrombotic microangiopathy.

CASE 2

A 32-year-old male patient was hospitalized with fever of unknown origin. His medical history revealed a liver transplant due to Budd-Chiari syndrome secondary to polycythemia vera (Vaquez-Osler disease) 6 years before admission. The postoperative period was complicated by acute rejection on day 5 and a biliary leak after the removal of a Kehr drain on day 86. Cyclosporine, azathioprine, and low-dose corticosteroids were used as antirejection therapy. Because of progressive thrombocytosis (880.000/mm³), the patient also received a low dose of hydroxyurea (250 mg daily).

On admission, the patient was febrile and complained of abdominal discomfort without any organ-specific symptom. Clinical examination revealed a bloated abdomen with hepatosplenomegaly. Laboratory results showed severe normocytic normochromic anemia with mild thrombocytosis and a normal leukocyte count and differentiation. In addition, laboratory analysis showed moderate renal impairment (calculated glomerular filtration rate, 63 mL/minute), cholestatic liver function abnormalities, hyperbilirubinemia, hyperuricemia, and elevated lactate dehydrogenase and C-reactive protein (Table 1, patient 2).

Hepatosplenomegaly (liver, 20 cm; spleen, 28 cm) without significant intra-abdominal adenopathies was visualized by CT. Liver biopsy revealed sinusoidal infiltration with medium-sized lymphocytes with little cytoplasm and irregular basophilic nuclei, mostly CD45+ and CD3+ T-lymphocytes. There was no marked infiltration by eosinophils, endothelitis, or foamy macrophages suggesting acute or chronic rejection.

Because of signs of progressive hypersplenism, splenectomy was performed. An investigation of the spleen showed massive infiltration of the red pulp by medium-sized lymphocytes with folded nuclei and moderate condensed chromatin. Immunophenotyping showed TCRγδ+, CD3+, CD4−, CD5−, CD7−, CD8+, CD56+, and clonal gene rearrangements. As such, a diagnosis of HSγδTCL was made. Unfortunately, a chromosomal investigation was not performed.

Treatment with CHOP-21 (cyclophosphamide, doxorubicin, vincristine, and methylprednisone) was initiated. After the second cycle, the patient was urgently admitted because of septic shock with multiorgan failure due to invasive disseminated aspergillosis. Despite maximal supportive care, the patient died 2 months after diagnosis.

DISCUSSION

PTLD is the second most frequent malignancy after solid organ transplantation (up to 20%). The overall incidence of PTLD is 25- to 100-fold higher than that in the general population and depends on the type of organ.5 These organ-specific differences are attributed to the duration and intensity of immunosuppression and the number of transplanted donor lymphocytes. The lowest incidence is found in kidney and liver transplants (1%-3%), whereas the highest incidence is observed after small intestine or multiorgan transplantation (up to 33%).5–8 The World Health Organization has divided PTLD into 3 categories: early lesions, polymorphic PTLD, and monomorphic PTLD.9 The majority of monomorphic PTLD (>80%) originates from B-lymphocytes and is associated with EBV. EBV-negative patients who receive an organ from an EBV-positive donor have the highest risk of developing PTLD (20-75 times higher), typically within the first year after transplantation.5, 8, 10 Rarely, EBV-negative PTLD arising from T-lymphocytes or natural killer cells can occur several years after transplantation.1, 6, 10 Another risk factor for developing PTLD is the preoperative or postoperative administration of OKT3 (a monoclonal T-cell blocking antibody) or antithymocyte globulins, which are given to prevent acute rejection.11 Hepatitis C (HCV) was thought to be associated with a higher incidence of PTLD for a long time.11 However, in a recently published large retrospective cohort study, Morton et al.12 failed to identify hepatitis C infection as a major risk factor for the development of PTLD. On the other hand, patients undergoing liver transplantation because of alcoholic cirrhosis were found to have higher mortality without an increased risk of developing PTLD. In a single-center study, patients with PTLD after liver transplantation had a 5-year survival of 69%.8

As mentioned before, PTLD arising from T-lymphocytes is rare. T-lymphocytes are characterized by the presence of a TCR that consists of 2 transmembranary noncovalent-bond αβ- or γδ-heterodimers and 5 CD3 subunits.13–15 Within monomorphic PTLD, HSγδTCL is a rare lymphoproliferative disorder that originates from the γδ-lymphocytes.3, 15–17 These γδ-lymphocytes represent less than 5% of all circulating T-lymphocytes; larger proportions can be found in the lymph nodes, gastrointestinal mucosa, skin, and red pulp of the spleen (up to 15%) and in pathological conditions such as leprosy, leishmaniasis, malaria, infection with hepatitis B, hepatitis C, or human immunodeficiency virus, rheumatoid arthritis, celiac disease, and Behçet's disease.17–22

On the basis of the δ-protein of TCR, γδ-lymphocytes can be subdivided into Vδ1-lymphocytes and Vδ2-lymphocytes, with predominantly Vδ1-lymphocytes in the healthy population.20, 23 These interferon-γ–producing lymphocytes are clearly increased in infections with EBV, human immunodeficiency virus, and autoimmune diseases and have a TCR-independent cytolytic pathway based on degranulation of perforin-consisting granules.24 On the other hand, Vδ2-lymphocytes are clearly increased in all kinds of infections, giving rise to lymphadenopathies.20

An analysis of the δ-protein has revealed that most HSγδTCLs originate from Vδ1-lymphocytes, and this possibly explains the absence of lymphadenopathies.13, 15, 18, 25

Most data on HSγδTCL come from case reports and small series. Recently, 2 large series have been published, including 45 and 21 patients, respectively.3, 4 HSγδTCL has a predilection to develop in young, predominantly male adults. The disease usually manifests with splenomegaly, with or without liver enlargement, but nearly always without peripheral lymphadenopathies. In general, a thoracoabdominal CT scan evaluation and positron emission tomography scintigraphy disclose no mediastinal or retroperitoneal adenopathies. Even during disease progression, the lymphoma remains preferentially localized within the spleen, liver, and bone marrow, without lymph node enlargement. B-symptoms such as unexplained fever, weight loss, and night sweats are frequently present, as well as nonspecific clinical findings such as fatigue, arthralgia, myalgia, and abdominal pain.1, 3, 4, 14–32 HSγδTCL can lead to acute liver failure requiring urgent liver transplantation. Therefore, if no other etiology of liver failure can be found, HSγδTCL should be considered because aggressive T-cell lymphomas can reappear in the donor liver quickly.33

In HSγδTCL, variable degrees of hematological abnormalities may occur. Severe thrombocytopenia is usually the striking feature and will be associated with mild anemia and leukopenia in more than half of patients.3, 4, 30 The mechanism of pancytopenia remains unclear, but possible explanations include the presence of splenomegaly and the secretion of cytokines that suppress normal hematopoiesis (eg, interferon-γ) by neoplastic γδ T-cells.3, 34 In the transplantation setting, calcineurin inhibitor–induced thrombotic microangiopathy needs to be excluded (the presence of schistocytes on a peripheral blood smear followed, in case of doubt, by bone marrow aspiration).

Early diagnosis is crucial, so a high index of suspicion is needed, especially in the case of young patients, primary EBV infection, EBV mismatch, or previous administration of antithymocyte globulins or OKT3.5 A diagnostic algorithm is presented in Fig. 3.

Figure 3. Diagnostic algorithm. Abbreviations: ATG, antithymocyte globulin; CMV, cytomegalovirus; CT, computed tomography; EBV, Epstein-Barr virus; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; PET, positron emission tomography; PTLD, posttransplant lymphoproliferative disorder; TMA, thrombotic microangiopathy.

A definite diagnosis depends on liver biopsy, bone marrow examination, or a splenectomy specimen. Hepatic involvement seems to be a constant feature, regardless of the presence of liver function abnormalities. Typically, a pathological examination of the liver shows marked infiltration by small to medium monoclonal γδ-lymphocytes in the sinusoids, while the portal tracts are spared. Because these infiltrating cells are dividing frequently, mitotic figures can be found (Fig. 2). This sinusoidal infiltration has to be differentiated from recurrent autoimmune or viral hepatitis and EBV hepatitis. The infiltrating cells in autoimmune hepatitis are mainly plasma cells, whereas in recurrent viral hepatitis, there is a predominant polyclonal T-lymphocyte infiltrate, but it is mixed with B-lymphocytes, plasma cells, and macrophages. EBV hepatitis gives rise to the formation of rows of polyclonal lymphocytes, but the infiltration is less severe in comparison with HSγδTCL. On immunohistochemistry, EBV inclusions can be demonstrated. HSγδTCL can easily be confused with other proliferative disorders. The differential diagnosis (mainly based on immunophenotyping) includes large granular lymphocyte leukemia, aggressive natural killer cell leukemia/lymphoma, and CD8+ T-cell chronic lymphocytic leukemia.29, 34, 35

Fresh or frozen tissue samples are preferred over formalin-treated, paraffin-embedded samples. The embedding process gives rise to fragmentation of the sample, disrupting the specific intrasinusoidal infiltrating pattern. Formalin, an aqueous solution of formaldehyde, reacts with amino acids and causes intramolecular and intermolecular crosslinks. These modifications may affect the results of molecular analysis.36 Therefore, formalin-treated and paraffin wax–embedded specimens should not be used unless this is necessary.

In the case of splenectomy, the typical findings consist of hyperplasia of the red pulp with atrophy of the white pulp.3, 4, 16, 26, 30 The characteristic trophism to liver and spleen sinusoids is attributed to altered expression of adhesion molecules on the surface of these malignant γδ-lymphocytes.4, 32 According to the literature, bone marrow involvement can be found in approximately two-thirds of the patients at diagnosis.3 Additional analysis for the presence of the EBV genome (in situ hybridization) or the expression of EBV-encoded latent membrane protein-1 (by immunohistochemistry) is normally negative, although a number of EBV-positive cases have been described.21

In recent years, immunophenotyping and conventional cytogenetic analysis have become essential parts of the diagnosis. Usually, these malignant γδ-lymphocytes are CD2+, CD3+, CD4−, CD5−, CD7+, CD8−, and TCRγδ+.3, 4, 13, 15, 26, 28 The main characteristic is the absence of CD4 and CD8 (although CD8 expression might occur, as illustrated by case 2), with coexpression of CD16, CD56, or CD57 (all natural killer cell–associated antigens).29 Clonal TCR-gene rearrangements are frequently found.31, 34, 37 The most frequently found genetic abnormality is isochromosome 7q10, and this is most likely the primary genetic disorder.4, 14, 26, 28, 29, 37, 38 This isochromosome is considered the hallmark of HSγδTCL but lacks specificity because isochromosome 7q may also be found in other malignant hematological diseases.14, 25 During tumor progression, additional abnormalities such as trisomy 8 and loss of the Y chromosome may be found.14, 37 Immunophenotyping and cytogenetic analysis can provide confirmation of the diagnosis of HSγδTCL only in the case of suggestive pathological findings. Isolated TCR rearrangements are insufficient to conclude the presence of T-cell lymphoma. Therefore, pathological examination should be considered the keystone of diagnosis.

HSγδTCL is characterized by a very aggressive clinical course. Despite an initial satisfactory response to induction treatment in most patients, the long-term outcome is dismal, with many patients succumbing within 1 year after diagnosis (range, 8 months to 3 years).3, 10, 14, 28, 30, 32 In a multivariate analysis, poor performance status, monomorphic disease, and graft organ involvement were associated with poor outcome.10

A variety of therapeutic options have been explored, including splenectomy, a CHOP-like combination of cytotoxic regimens, purine analogues, monoclonal antibodies, and autologous and allogeneic hematopoietic stem cell transplantation. Unfortunately, no treatment option seems to improve life expectancy,3, 37 although allogeneic stem cell transplantation seems to be beneficial.39 Currently, there is insufficient evidence of beneficial effects of antiviral therapy.40

As demonstrated by the presentation of the second case, chemotherapy has a poor outcome because of the high treatment-related mortality (estimated to be approximately 50%) by infectious complications or multiple organ failure. As demonstrated by the first case report, allogeneic transplantation may result in complete remission (even in chemotherapy-refractory cases), but managing graft-versus-host disease targeting the allogeneic liver appears to be a tremendous challenge. Treatment with monoclonal antibodies against CD20 (pan-B-cell) has low treatment-related morbidity and mortality rates and can result in complete remission in posttransplant B-cell lymphomas.41, 42 However, none of these therapies is useful in T-cell PTLD. Therefore, at present, cytotoxic chemotherapy and/or allogeneic stem cell transplantation must be considered the only therapeutic options for T-cell PTLD.

In conclusion, both patients were good examples of the origin and evolution of HSγδTCL. They were young adult liver transplant recipients whose posttransplant courses were complicated by several episodes of rejection requiring profound immunosuppressive therapy, which increased the risk of PTLD. Both patients were hospitalized because of fever and hepatosplenomegaly without peripheral lymphadenopathies. Pathological examination demonstrated the presence of sinusoidal infiltration of the liver and spleen by typical γδ-lymphocytes.

The characteristic trophism, attributed to an altered expression of adhesion molecules, was not influenced by the presence of an allogeneic liver, and this suggests that malignant γδ-lymphocytes express universal nonspecific adhesion molecules because these lymphocytes infiltrate both the autologous spleen and allogeneic liver. Another possible explanation is that the adhesion molecules of the donor and receptor are identical because of the human leukocyte antigen match.

Despite adequate therapy (several lines of combination chemotherapy followed by sibling hematopoietic stem cell transplantation and CHOP, respectively), both patients died within 1 year after diagnosis because of treatment-related complications.