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Hepatosplenic T-cell lymphoma diagnosed using flow cytometry. A single-center study of 12 cases from North India

 Department of Hematology, SGPGI, Lucknow, Uttar Pradesh, India

Date of Submission18-Oct-2020
Date of Decision16-Dec-2020
Date of Acceptance12-Apr-2020
Date of Web Publication23-Oct-2021

Correspondence Address:
Ruchi Gupta,
Department of Hematology, SGPGI, I Block, Hematology Building, Lucknow, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_877_19

 > Abstract 

Background: Hepatosplenic T-cell lymphoma (HSTCL) is a rare fatal T-cell neoplasm with unique clinical and laboratory features. There is, however, significant morphological and immunophenotypic heterogeneity which may lead to diagnostic dilemma.
Aims and Objectives: The study was aimed to study the prevalence and clinic-pathological spectrum of this rare variant of T cell lymphoma in the Indian subcontinent.
Material and Methods: A retrospective analysis of all consecutive cases of HSTCL diagnosed over a period of 6 years was carried out. The clinical and laboratory parameters of all these patient were reviewed and analysed.
Results: A total of 12 cases of HSTCL were diagnosed during this period which accounted for 1.76% of all non-Hodgkin's lymphomas (NHLs) and 9.1% of all T-cell NHLs. The median (range) age of presentation was 23 (16–30) years.Leukocytosis, peripheral blood (PB) involvement, and a blastic morphology were noted in 41%, 67%, and 58% of the cases, respectively. FCI proved these cells to have a mature, dual-negative (CD4−/CD8−) T-cell phenotype with a gamma–delta T-cell receptor restriction. Frequent loss of CD5 expression (84%) was also noted. These patients invariably had a fatal outcome and majority died within a year of diagnosis.
Conclusion: The incidence of leukocytosis and a blastoid morphology is quite frequent in HSTCL. Hence, a differential diagnosis of HSTCL should always be considered in young patients presenting with splenomegaly and exhibiting atypical lymphoid/blastoid cells in the PB or a marrow. An FCI can readily diagnose and differentiate them from an acute lymphoblastic leukemia/lymphoma.

Keywords: Hepatosplenic T cell Lymphoma, India, Flow Cytometry

How to cite this URL:
Rahman K, Gupta T, Gupta R, Singh L, Chandra D, Sarkar MK, Singh MK, Kumar S, Nityanand S. Hepatosplenic T-cell lymphoma diagnosed using flow cytometry. A single-center study of 12 cases from North India. J Can Res Ther [Epub ahead of print] [cited 2022 Aug 16]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=329056

 > Introduction Top

Hepatosplenic T-cell lymphoma (HSTCL) is an uncommon subtype of peripheral T-cell originating from the restricted gamma–delta (γδ) or less frequently alpha–beta (αβ) T-cell receptor (TCR) cytotoxic memory T-cells. It represents ~ 1%–2% of all non-Hodgkin's lymphomas (NHLs).[1] Clinically, it is characterized by hepatosplenomegaly, absent or minimal lymphadenopathy, and peripheral blood (PB) cytopenias in young adults. HSTCL has a rapidly progressive clinical course with dismal outcome. The reported median survival is less than a year, and the 5-year overall survival rate is about 10%–15% only.[2],[3] Classically, the tumor cells have a monomorphic small-to-medium size, which preferentially infiltrate hepatic, splenic, and bone marrow (BM) sinusoids. BM is reportedly involved in nearly all the patients.[1],[4],[5] Morphological variation in the form of blastic transformation during the terminal phase of the disease has been reported but is uncommon.[6] Immunophenotypically, these cells are mature CD3 + and CD2 + T-cells, which are dual negative for CD4 and CD8 and express clonally restricted γδ TCR or less frequently αβ TCR.

A retrospective analysis of 12 patients of HSTCL diagnosed on flow cytometric immunophenotyping (FCI) of the BM aspirate/PB samples is being presented in this article.

 > Materials and Methods Top

A retrospective analysis of all the consecutive patients of HSTCL diagnosed through flow cytometry over a 6-year period (July 2013 to June 2019) was carried out. Relevant clinical history along with hematological and biochemical profiles of all the patients was retrieved from hospital electronic medical records.

For morphological evaluation, PB smears, BM aspirates, and biopsies stained with Leishman, May–Grunwald–Giemsa, and hematoxylin and eosin stains, respectively, were reviewed.

FCI using 4–8 color panels were performed on PB or BMA samples. A stain-lyse-wash protocol was used with following monoclonal antibodies: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD34, CD38, CD45, CD56, CD200, FMC7, αβ TCR, γδ TCR, kappa light chains, and lambda light chains in different tube combinations. Briefly, 100–300 μl of BMA/PB samples was incubated with antibody cocktails for 20–30 min in dark. It was then washed with phosphate-buffered saline to remove the excess of unbound antibodies. This was followed by red cell lysis using commercial lysing agents, followed by wash and a final suspension of cell pellet in paraformaldehyde. The processed samples were acquired on a BD FACS Canto II platform with FACS Diva software (BD Biosciences, San Jose, CA, USA), which was also used for the analysis of samples.

 > Results Top

Approximately 681 patients of NHL were diagnosed during this time period, of which 131 were T-cell and 550 were B-cell NHLs. There were 12 patients of HSTCL, which accounted for1.76% of all NHLs and 9.16% of all T-cell NHLs. The median (range) age of presentation was 23 (16–30) years, with a definite male preponderance. The summary of clinic-pathological features is shown in [Table 1]. The details of individual patients are shown in [Supplementary Table 1].
Table 1: Clinical and laboratory features of patients cohort

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Marrow infiltration was seen in all the patients with a variable morphology. Seven (58.3%) of these patients showed morphology simulating blasts, whereas the rest five displayed atypical lymphoid cell infiltration. A single case mimicked monocytoid blasts with moderate-to-abundant cytoplasm, presence of cytoplasmic vacuoles, folded nuclear contour, opened up chromatin, and 1–2 prominent nucleoli. BM biopsy was available in 11 patients. The classical sinusoidal pattern of involvement was noted in 5 (45.5%) patients, whereas 6 (54.5%) patients show diffuse infiltration. [Figure 1] shows the morphological features of few representative cases.
Figure 1: Morphological spectrum of gamma–delta hepatosplenic T-cell lymphoma, first row showing the classical pattern with small-to-medium-sized lymphoid cells and sinusoidal pattern of infiltration, second row showing the presence of blastic cells and a diffuse replacement of the marrow spaces on bone marrow biopsy

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On FCI, the atypical cells were seen in the bright CD45 region and expressed CD3 in 11 (91.7%) patients. CD3 was negative in one case. The frequency of CD2, CD7, and CD5 expression was 100%, 91.7%, and 16.6%, respectively. Ten (81.8%) patients were dual negative for CD4 and CD8, and rest of the two patients were CD4+/CD8 − and CD4−/CD8+, respectively. Antibodies against surface receptor αβ and γδ were evaluated in seven patients, all of which were γδ TCR positive. Of the five cases, where the TCR could not be tested, two cases had a typical sinusoidal pattern of marrow involvement, whereas three had diffuse marrow infiltration. All these had the presence of hepatosplenomegaly and CD4/CD8 dual-negative mature T-cell phenotype. Based on the clinical features, pattern of marrow involvement, and available immunophenotyping, HSTCL was the best possible diagnosis. CD56 could be applied in nine patients only and were positive in five (55.5%) patients. The scatter plots of one of the representative cases with CD56 expression are shown in [Figure 2]. Immaturity markers such as CD34, TdT, and CD99 were negative in all the cases. None of the patients expressed any aberrant myeloid or B-cell antigen. Conventional karyotyping was done in five patients. Four patients showed a normal karyotype, whereas one showed a complex karyotype, which included trisomy 8 and del 7q. Limited data were available on the treatment and follow-up. Different protocols, such as cyclophosphamide, doxorubicin hydrochloride, Oncovin, and prednisone; hyper-cyclophosphamide, vincristine, adriamycin, and dexamethasone; and etoposide, solumedrol, high-dose cytarabine (ara-C), and cisplatin, were used in six patients. Three of them died within 9 months of diagnosis, whereas the other three were lost to follow-up.
Figure 2: Immunophenotypic profile of one of the cases which expressed CD56 with slightly dimmer CD3 expression. It was positive for CD2 and CD7 (not shown), dual negative for CD4 and CD8, and negative for CD5 and expressed gamma–delta T-cell receptor. It was negative for all immaturity markers

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 > Discussion Top

HSTCL was described for the first time in 1981 by Kadin et al. under the designation of “erythrophagocytic T-gamma lymphoma.”[7] The term “HSTCL” was first used in 1990 to describe two patients with hepatosplenic presentation, sinusoidal tropism, and a γδ phenotype.[8] This was later included as a type of peripheral T-cell lymphoma in the WHO classification of hematolymphoid neoplasm. The reported incidences are 3.0%, 2.3%, and 0.2% in North America, Europe, and Asia, respectively.[3] The incidence of HSTCL, as reported from India in two other studies, is 0.6% of all NHLs and 4.2% of mature T-NHL,[5] whereas it accounted for ~ 1.76% of all lymphomas and 9.16% of all T-NHLs in our case series. Most of the patients have an unknown etiology. However, about 10%–20% of the patients exhibit features of immune dysregulation in the form of autoimmune disease or a posttransplant setup.[9] None of the patients in our series had any evidence of previous autoimmune or inflammatory disorder.

The median age of presentation in our cohort was 23 years with male predominance (M: F ratio: 2:1). This was almost a decade less than the data reported in Western literature.[1],[4],[5],[10] One of the Chinese studies had a similar median age of presentation as ours.[11] HSTCL presenting with leukocytosis, which is uncommonly described, can be confused with acute leukemia.[1],[6] In contrast, 41.6% of our patients had leukocytosis at presentation. Circulating atypical cells were noted in two-third (67%) of our patients, which was similar to those reported by other authors. A comparison of clinical and laboratory features between different studies is shown in [Table 2].
Table 2: Comparison of clinical features among different series published in English literature

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BM involvement, although often subtle, is almost always present in HSTCL. In the early stages, malignant cells have a sinusoidal distribution, are monotonous, intermediate sized with rim of pale cytoplasm, loosely condensed chromatin, and inconspicuous nucleolus.[11],[14] However, they may vary in morphology from case to case and show different patterns of involvement. Sinusoidal pattern of infiltration is classically described, however, interstitial alone or interstitial along with sinusoidal pattern has also been reported. With disease progression, the pattern of BM involvement may increasingly become interstitial and/or diffuse along with transition in morphology, i.e., presence of larger and blastic cells.[6],[14] Unusual morphological patterns mimicking metastatic marrow infiltration have also been reported.[5] One of our patients displayed large cells with round to slightly indented nucleus and moderate-to-abundant cytoplasm with very fine granules, mimicking monoblastic leukemia. A higher prevalence of leukemic presentation and cases with blastic morphology in the present study may be attributed to a referral bias because of our center being a tertiary care center. By the time patients seek management from our center, there seems to be a noticeable disease progression. Hence, it is imperative for a hematopathologist to keep in mind these unusual characteristics of HSTCL while interpreting patients with organomegaly and blast-like cells in the PB and marrow. In these settings, acute leukemia and blast crisis in chronic myeloid leukemia are more likely differentials. FCI clinches the diagnosis by ascertaining the mature T-cell phenotype in these cases. These cells typically express bright CD45 and are positive for surface CD3, CD7, and CD2 while being negative for all immaturity markers such as CD34/TdT/CD99/CD1a. These are usually dual negative for CD4 and CD8 and frequently show loss of CD5 expression. The γδ TCR is positive in majority of patients and rarely expresses αβ TCR. In the present cohort, all the seven patients tested for TCR expressed γδ TCR. T-cell acute lymphoblastic leukemia (ALLs) with γδ TCR expression is another close mimicker of HSTCL. The former usually show a moderate-to-dim expression of CD45, dimmer, and variable expression of surface CD3 along with at least one of the immaturity marker (CD99/TdT/CD34/CD1a). In contrast, HSTCLs brightly and homogeneously express CD45 and surface CD3 and are negative for the immaturity markers. In addition, the presence of a characteristic sinusoidal pattern of marrow involvement and the presence of isochromosome 7 on FISH analysis can readily distinguish these two entities.

An unusual case of surface CD3-negative HSTCL has also been reported in the literature.[15] We also noted a similar case in a 22-year-old male patient, who presented with pancytopenia and splenomegaly, with typical sinusoidal pattern of involvement in BM biopsy. The absence of all immaturity markers, with the presence of typical clinical and morphological features, favored this case as HSTCL over a T-cell ALL. Antibodies against γδ TCR are usually not a part of primary panel of antibodies in many of the laboratories. However, it is suggested that in cases with blastic morphology of cells, presence of surface CD3, and absence of immaturity markers, possibility of HSTCL should be ruled out by addition of these antibodies in a an appropriate clinical setting. A comparative table of the immunophenotypic findings from some of the published studies is summarized in [Table 3].
Table 3: Immunophenotypic characteristic of hepatosplenic T-cell lymphoma - comparison between studies

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Clinically, HSTCL is an aggressive subtype of extranodal lymphoma with hepatosplenic presentation. It has a dismal treatment outcome with high mortality.[5],[11] In the present study, 50% of the patients who opted for treatment (n = 6) died within 9 months of diagnosis, reiterating the poor prognosis of this disease.

 > Conclusion Top

HSTCL is an uncommon group of NHL with a unique clinical, morphological, and immunophenotypic characteristic. These patients can present with leukocytosis and/or blastic morphology, thus mimicking an ALL. FCI is mandatory for establishing a correct diagnosis and initiating management accordingly.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

Belhadj K, Reyes F, Farcet JP, Tilly H, Bastard C, Angonin R, et al. Hepatosplenic gammadelta T-cell lymphoma is a rare clinicopathologic entity with poor outcome: Report on a series of 21 patients. Blood 2003;102:4261-9.  Back to cited text no. 1
Durani U, Go RS. Incidence, clinical findings, and survival of hepatosplenic T-cell lymphoma in the United States. Am J Hematol 2017;92:E99-101.  Back to cited text no. 2
Vose J, Armitage J, Weisenburger D; International T-Cell Lymphoma Project. International peripheral T-cell and natural killer/T-cell lymphoma study: Pathology findings and clinical outcomes. J Clin Oncol 2008;26:4124-30.  Back to cited text no. 3
Falchook GS, Vega F, Dang NH, Samaniego F, Rodriguez MA, Champlin RE, et al. Hepatosplenic gamma-delta T-cell lymphoma: Clinicopathological features and treatment. Ann Oncol 2009;20:1080-5.  Back to cited text no. 4
Patkar N, Nair S, Alex AA, Parihar M, Manipadam MT, Arora N, et al. Clinicopathological features of hepatosplenic T cell lymphoma: A single centre experience from India. Leuk Lymphoma 2012;53:609-15.  Back to cited text no. 5
Visnyei K, Grossbard ML, Shapira I. Hepatosplenic γδ T-cell lymphoma: An overview. Clin Lymphoma Myeloma Leuk 2013;13:360-9.  Back to cited text no. 6
Kadin ME, Kamoun M, Lamberg J. Erythrophagocytic Tγ lymphoma. New Engl J Med 1981;304:648-53.  Back to cited text no. 7
Farcet JP, Gaulard P, Marolleau JP, Le Couedic JP, Henni T, Gourdin MF, et al. Hepatosplenic T-cell lymphoma: Sinusal/sinusoidal localization of malignant cells expressing the T-cell receptor γδ. Blood 1990;75:2213-9.  Back to cited text no. 8
Thai A, Prindiville T. Hepatosplenic T-cell lymphoma and inflammatory bowel disease. J Crohns Colitis 2010;4:511-22.  Back to cited text no. 9
Macon WR, Levy NB, Kurtin PJ, Salhany KE, Elkhalifa MY, Casey TT, et al. Hepatosplenicalphabeta T-cell lymphomas: A report of 14 cases and comparison with hepatosplenic gamma delta T-cell lymphomas. Am J Surg Pathol 2001;25:285-96.  Back to cited text no. 10
Lu CL, Tang Y, Yang QP, Wang M, Zhao S, Bi CF, et al. Hepatosplenic T-cell lymphoma: Clinicopathologic, immunophenotypic, and molecular characterization of 17 Chinese cases. Hum Pathol 2011;42:1965-78.  Back to cited text no. 11
Weidmann E. Hepatosplenic T cell lymphoma. A review on 45 cases since the first report describing the disease as a distinct lymphoma entity in 1990. Leukemia 2000;14:991-7.  Back to cited text no. 12
Yabe M, Medeiros LJ, Tang G, Wang SA, Ahmed S, Nieto Y, et al. Prognostic factors of hepatosplenicT-cell lymphoma (HSTCL): A clinicopathologic, immunophenotypic, and cytogenetic analysis of 28 patients. Am J Surg Pathol 2016;40:676-88.  Back to cited text no. 13
Vega F, Medeiros LJ, Gaulard P. Hepatosplenic and other gammadelta T-cell lymphomas. Am J Clin Pathol 2007;127:869-80.  Back to cited text no. 14
Chauhan R, Tyagi S, Mirgh S, Mishra P, Seth T, Mahapatra M, et al. Expect the unexpected – Loss of surface CD3 on flow cytometry in hepatosplenic T-cell lymphoma: An eye opener. Indian J Pathol Microbiol 2018;61:275-7.  Back to cited text no. 15
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