Yen-Chuan Hsieh

A comparative study of flow cytometric T cell receptor Vβ repertoire and T cell receptor gene rearrangement in the diagnosis of large granular lymphocytic lymphoproliferation

Large granular lymphocytes (LGLs) are medium‐ to large‐sized lymphocytes with azurophilic cytoplasmic granules. Reactive vs. neoplastic LGLs are usually morphologically indistinguishable.

The spectrum of T-cell and natural killer/T-cell neoplasms with leukaemic presentation in a single institution in Taiwan

T cell and natural killer (NK)/T‐cell neoplasms are rare and may occasionally present as leukaemia. We retrospectively searched T cell and NK/T‐cell tumours in a single institution in Taiwan from January 2000 to December 2009 and identified 137 (19.1%) patients with T cell and NK/T‐cell tumours among 718 patients with lymphoid neoplasms. Among these 137 patients, 18 (13.1%) presented with leukaemia including T‐lymphoblastic lymphoma/leukaemia (T‐LBL), T‐cell prolymphocytic leukaemia, aggressive NK‐cell leukaemia, adult T‐cell lymphoma/leukaemia (ATLL), T‐cell large granular lymphocytic (T‐LGL) leukaemia and unspecified peripheral T‐cell lymphoma. Cases with concurrent lymphoma, higher absolute leukaemic cell counts and elevated lactate dehydrogenase level carried a poorer prognosis. The survival was dichotomous, with a very poor prognosis for patients with T‐LBL, T‐cell prolymphocytic leukaemia, aggressive NK‐cell leukaemia, ATLL in acute phase and unspecified peripheral T‐cell lymphoma, while those with T‐LGL leukaemia and ATLL in chronic phase had a favourable outcome.

Adult T-cell leukemia/lymphoma comprising non-floral leukemic cells in Taiwan, a country non-endemic for human T-cell leukemia virus type I

Lymphocytosis is usually the first hint of chronic lymphoproliferative disorders, most commonly chronic lymphocytic leukemia. However, as there are overlapping morphological features in various entities of chronic lymphoproliferative disorders, ancillary studies such as flow cytometry and occasionally molecular techniques are needed to reach a definitive diagnosis. Here, we present a patient with mild lymphocytosis comprising mature small leukemic cells devoid of deeply indented or nuclear lobulation. Subsequent laboratory studies confirmed a chronic phase of human T-cell lymphotropic virus type I (HTLV-I) associated adult T-cell leukemia/ lymphoma (ATLL). A 57-year-old Taiwanese male presented, in March 2009, with a history of leukemia recently diagnosed at other hospital. The hemogram showed a white blood cell count of 14.66 10/L with 49.1% lymphocytes and a normal hemoglobin and platelet count. The serum levels of calcium and lactate dehydrogenase (LDH) were normal. There were no B symptoms, lymphadenopathy, or hepatosplenomegaly. A blood smear film showed small to mediumsized abnormal lymphocytes with condensed chromatin [Figures 1(A)–1(C)]. There was no significant nuclear lobulation to suggest floral cells or flower cells. Flow cytometry showed that these gated lymphocytes expressed CD2, CD3, CD4, CD5, and CD25 but not CD8, CD20, CD38, or CD56. Furthermore, there was an aberrant loss of CD7 expression [Figure 1(D) and 1(E)]. Clonality study for T-cell receptor gamma chain gene rearrangements using polymerase chain reaction method showed clonal results. Serum anti-human T-cell leukemia virus (HTLV)-I/II was positive. Using Southern blot analysis, the DNA extracted from peripheral blood leukocytes and digested with EcoRI was positive for HTVL-I proviral integration with an extraordinary single band smaller than 9 kb indicating a defective provirus [Figure 1(F)]. The final diagnosis was ATLL in chronic phase according to the criteria of the Japanese Lymphoma Study Group [1]. Interestingly, the serum of the patient’s spouse was positive for anti-HTLV-I/II, whereas the hemogram was normal without atypical lymphocytes. HTLV-I infection is endemic in Japan, Africa, South America, Caribbean basin, Southern part of North America, and Eastern Europe [2]. Most HTLV-I-infected individuals remain asymptomatic carriers, whereas 1–5% of those infected carry a lifelong risk of developing ATLL [2]. Taiwan is a country non-endemic for HTLV-I with the seropositive rate among blood donors at 0.06% [3] and very few cases of ATLL [4,5]. Morphologically, a broad range of cytological features may be present in ATLL with the most characteristic findings of leukemic cells with nuclear lobulation, the so-called floral cells or flower cells [6,7]. In the previous reports of ATLL in Taiwan, all the cases with leukemic phase exhibited

T-cell large granular lymphocytic leukemia with pleomorphic nuclei and colonic infiltration with chronic diarrhea.

Large granular lymphocytes (LGLs) in the peripheral blood (PB) represent either cytotoxic T-cells or natural killer (NK) cells. T-cell LGL (T-LGL) proliferations range from reactive conditions frequently associated with autoimmune diseases to frank leukemia [1]. T-LGL leukemia has been defined by the 2008 World Health Organization (WHO) classification as a heterogeneous and indolent disorder characterized by a persistent (46 months) increase in the PB LGLs, usually between 2 and 206 10/L, without a clearly identified cause [2]. Patients may be either asymptomatic or with fever and clinical symptoms related to infections. The major hematological findings are neutropenia, anemia, and, less commonly, thrombocytopenia. The most common sites of leukemic infiltration are bone marrow (BM), liver, and spleen, and occasionally lymph node (LN) [1,3,4]. To our knowledge there has been no report on colonic infiltration by TLGL leukemia. We report such a case with a longterm follow-up period. A 29-year-old male presented with intermittent fever and diarrhea several times a day in April 1995. Biopsies of the BM, liver, and colon revealed interstitial infiltration by atypical lymphocytes consistent with LGL leukemia. The patient received medication with prednisolone; however, diarrhea persisted for years, during which he had mild anemia (hemoglobin range: 9.3–12.7 g/dL) and normal white blood cell (WBC) and platelet counts. Annual colonoscopic examination from 2004 to 2009 revealed diffuse colonic ulcers with mucosal swelling and easy-touch bleeding from the splenic flexure to the rectum. An episode of pneumonia occurred in December 2006, which lead to bi-lobectomy of the right middle and lower lobes. Abdominal computed tomography (CT) in May 2008 revealed generalized thickening of the colon wall and lymphadenopathy in the abdomen and bilateral inguinal LNs. Repeat CT in December 2009 revealed a slight enlargement of the nodes as compared to that in 2008, and cyclophosphamide was prescribed in addition to prednisolone. Two months later in February 2010 his long-term diarrhea finally resolved. Histopathology of the annual colonic biopsies revealed similar features of atypical small lymphocytic infiltration in the lamina propria, with relatively intact colonic glands except for focal erosion and ulceration (Figure 1). These small, atypical lymphocytes exhibited scanty to moderate cytoplasm, irregular nuclear shape, and infrequent mitoses without lymphoepithelial lesions [Figure 1(B)]. Immunohistochemically, the tumor cells expressed CD2, CD3, CD8, and T-cell intracellular antigen, but not CD4, CD20, CD30, CD56, and CD57 [Figures 1(C)–1(E)]. The expression of CD5, CD7, CD16, and granzyme B was partial. The proliferation fraction as determined by Ki-67 immunostaining was low. In situ hybridization for Epstein–Barr virus-encoded mRNA was negative. The first flow

CD4 and CD8 double-negative adult T-cell leukemia/lymphoma with monomorphic cells expressing CD99: a diagnostic challenge in a country non-endemic for human T-cell leukemia virus.

Adult T‐cell leukemia/lymphoma (ATLL) is a peripheral T‐cell neoplasm caused by human T‐cell lymphotropic virus type I (HTLV‐I). The neoplastic cells are highly pleomorphic and are usually CD4+ and CD8− phenotypically. We reported the case of a 46‐year‐old woman presenting with fever, abdominal distention, lymphadenopathy, leukocytosis and hypercalcemia. Nodal biopsy showed diffuse infiltration by monomorphic small to medium‐sized atypical lymphocytes expressing CD3, CD25, CD30 and CD99, but not CD1a, CD4, CD8, CD34, terminal deoxynucleotidyl transferase or ALK. An initial diagnosis of T‐lymphoblastic leukemia/lymphoma was made based on cytomorphology, CD4 and CD8 double negativity, and the expression of CD99. The diagnosis was later revised to ATLL based on the positive serology study for anti‐HTLV I/II antibody and confirmation by the clonal integration of HTLV‐I proviral DNA into the tumor tissues by Southern blotting analysis. The patient had a stage IVB disease and died of septic shock after 2 courses of chemotherapy 3 months after diagnosis. Immunohistochemical staining for CD99 in archival ATLL tissues showed a positive rate of 67% (4 of 6 tumors). Our case showed that ATLL with atypical morphology and immunophenotype in HTLV non‐endemic areas might pose a diagnostic challenge and CD99 expression is frequent in ATLL.

Colonic CD30 positive plasmablastic plasmacytoma masquerading as anaplastic large cell lymphoma

Sir, Solitary plasmacytomas are single localised tumours of monotypic plasma cells without features of plasma cell myeloma/multiple myeloma (MM), nor physical or radiographical evidence of additional plasma cell tumours. Solitary plasmacytomas may occur in the bones or in extraosseous (extramedullary) tissues, with around 75% of extramedullary plasmacytoma occurring in the upper respiratory tract and very rare cases in the gastrointestinal tract. Morphologically, neoplastic plasma cells may range from mature, atypical, immature to plasmablastic forms. In a prior study of MM by Greipp et al., plasmablastic morphology accounted for around 8% cases and was associated with more aggressive disease and a shortened survival. Anaplastic large cell lymphomas (ALCLs) are mature Tcell lymphomas comprising anaplastic cells expressing CD30 and are classified as ALK positive or ALK negative ALCLs depending on ALK gene rearrangement and protein expression. In our recent study of primary gastrointestinal ALCL, we identified a case of CD30 positive extramedullary plasmacytoma of the plasmablastic morphology in the colon, erroneously diagnosed as ALCL initially. The patient subsequently developed malignant pleural effusion and leukaemic transformation. In this report, we present the pertinent clinicopathological features and discuss the diagnostic pitfalls. A 72-year-old male presented with abdominal distention and bloody diarrhoea for a week in March 2012. He also had nausea, vomiting, weakness with poor appetite and body weight loss. His ECOG performance status score was 4. Abdominal computed tomography (CT) scans revealed a soft tissue mass, 7.5 7.7 9.1 cm, in the caecum extending to the ascending colon and ileocaecal valve, and small nodules in the peritoneal cavity with ascites. In addition, there were multiple hypodense lesions in the liver and numerous enlarged lymph nodes in the mesentery and para-aortic regions. Laboratory examination revealed mild leukocytosis (10.9 10/mL) without leukaemic cells, mild anaemia (haemoglobin level 11.8 g/dL), and normal platelet count. His serum levels of LDH (657 IU/L; reference 85e227) and b2-microglobulin (5090 mg/L) were both elevated, while that of calcium and creatinine were within normal ranges. His albumin level was 1.8 g/dL. Colonoscopy revealed a 5 cm nodular lesion in the transverse colon. Section of the colonic biopsy revealed sheets of large neoplastic cells infiltrating in the lamina propria. The tumour was diagnosed as ALK negative ALCL based on the anaplastic large cells morphology (Fig. 1), with the expression of CD7, CD30, and CD45 (partial) but not ALK or EBV (in situ hybridisation for EBV-encoded mRNA or EBER). Pleural effusion cytology was diagnosed as malignant, most likely lymphoma. Bone marrow biopsy was negative. A stage IV disease with IPI score of 5 was diagnosed. He was treated

Effusion-based lymphoma with morphological regression but with clonal genetic features after aspiration

Effusion‐based lymphoma (EBL) is a rare but distinct entity of large B‐cell lymphoma in effusion without association with human herpes virus‐8 (HHV‐8). Spontaneous regression after pleurocentesis has been observed; but to our knowledge, there are no reports on the morphological and molecular features of subsequent aspirations in regressing cases. Here, we report the case of a 92‐year‐old male with chronic obstructive pulmonary disease, who presented with right pleural effusion. He had no human immunodeficiency virus, hepatitis B virus, or hepatitis C virus infection, and CT scans revealed no mass lesion. The first pleural effusion aspiration cytology revealed large lymphoma cells with vesicular nuclei, irregular nuclear contours, and prominent nucleoli, consistent with EBL. The second aspiration cytology showed a few slightly enlarged lymphocytes in a background of small lymphocytes. Immunohistochemical study on cell block of the second aspiration revealed equal amounts of CD3‐positive and CD20‐positive cells. All these cells on the section tested negative for HHV‐8 through immunohistochemistry and Epstein‐Barr virus by in situ hybridization. Our initial impression was EBL in regression. However, flow cytometric immunophenotyping showed monotypic light chain expression of the gated B‐cells. B‐cell receptor gene rearrangement study showed a clonal result. Furthermore, fluorescence in situ hybridization revealed rearrangement of IGH gene. The diagnosis of the second aspiration was EBL with morphological regression but retained clonal genetic features. The patient passed away one month after diagnosis without chemotherapy. This case illustrated the importance of ancillary studies in confirming the clonal nature of a morphologically regressing EBL.

Primary splenic low-grade follicular lymphoma presenting with leukaemia and large cell transformation in the marrow

previous reports of thyroid PTCL-NOS, some had an aggressive course with the patient succumbing within 5e13 months despite chemotherapy and radiotherapy. However, others reported spontaneous regression with subsequent disease-free survival of up to 97 months. In this case, the decision was made for observation only due to the low disease burden. In conclusion, this is a rare case of thyroid PTCL-NOS arising in a Caucasian male. Histologically, the features were like that of extranodal marginal zone lymphoma. Immunohistochemistry, flow cytometry and molecular studies were essential to clinching the diagnosis. It is yet to be elucidated if thyroid T-cell lymphoma is a distinct entity amongst the PTCLs-NOS. The natural history of reported thyroid T-cell lymphoma is variable and limited data are available on prognosis and treatment.

Aggressive natural killer-cell neoplasm presenting in the marrow: a report of two cases including one with gains of chromosomes 4q and 9p

Aggressive nature killer (NK)-cell neoplasm includes aggressive NK-cell leukemia (ANKL) and extranodal NK/T-cell lymphoma (ENKTL), nasal type. ANKL is rare and is characterized by a systemic neoplastic proliferation of NK-cells, usually with a leukemic presentation. ENKTL is a predominantly extranodal lymphoma, occurring mainly in the upper aerodigestive tract. Both are aggressive neoplasms strongly associated with Epstein-Barr virus (EBV). Here we report two patients with aggressive NK-cells neoplasms localized in the bone marrow (BM) who presented as prolonged fever, anemia, and thrombocytopenia. Both were treated initially as infectious disease. Imaging studies revealed splenomegaly without any nodular lesion or lymphadenopathy. BM examination revealed extensive involvement by EBV-positive NK-cells in both cases. Staging workup including nasal examination/biopsy was negative. Both patients passed away in a month. One case showed gains of chromosomes 4q and 9p by array comparative genomic hybridization. Both tumors were diagnostically challenging due to the unusual clinical presentation and absence of leukemic change, tumor mass or lymphadenopathy. Our cases demonstrate that lymphoma should be considered in patients with fever of unknown origin and bone marrow aspiration/biopsy should be performed as early diagnosis and novel therapeutic regimens may benefit these patients.

Clonal lymphoproliferation of T cell large granular lymphocytes with pleomorphic nuclei following mantle cell lymphoma

Large granular lymphocytes (LGLs) are mediumto largesized lymphocytes with azurophilic cytoplasmic granules serving as the main effector cells of cell-mediated cytotoxicity of either T or natural killer (NK) cell origin. T cell LGL lymphoproliferation may be reactive or neoplastic; and in most instances, the morphology of the leukemic cells in T cell LGL leukemia (T-LGLL) is indistinguishable from that of the normal LGLs. Here, we present a case of clonal T-LGL lymphoproliferative disorder (LPD) exhibiting low LGL counts, but marked nuclear atypia with pleomorphic nuclei. A 60-year-old male presented with fever, chills, and general weakness 2 days after the third dose of a monthly maintenance therapy with rituximab in July 2012. He was in nearly complete remission after five courses of HyperCVAD chemotherapy for mantle cell lymphoma (MCL). The patient’s history showed a diagnosis with MCL in May 2011 via a nodal biopsy that showed a nodular growth pattern with classic cytomorphology and neoplastic cells expressing CD5, CD20, CD43, IgD, IgM, bcl-2, and cyclin D1, but not CD3, CD10, CD23, or bcl-6. At that time, flow cytometric immunophenotyping confirmed peripheral blood involvement with a phenotype of CD5 CD19 CD20 CD10 CD23 and monotypic surface lambda light chain expression. Fluorescence in situ hybridization assays with dual color break apart probes showed that the lymphoma cells were positive for IGH and CCND1 rearrangements, but without aberrations at the BCL2, BCL6, or MYC loci. Although we did not have an IGH/CCND1 dual color dual fusion probe, we believed our case to be the result of a reciprocal translocation involving IGH and CCND1 loci. Laboratory data in July 2012 showed Hb at 12.7 g/dL, WBC count of 1,300/lL including 34 % LGLs with an absolute count of 442/lL and a platelet count of 20,000/lL. The LGLs included two populations: one with normal morphology [absolute count at 65/lL (14.7 %)] and the other with abnormal morphology [absolute count at 377/lL (85.3 %)] showing marked nuclear irregularity (Fig. 1), resembling flower cells as seen in adult T cell leukemia/lymphoma. Flow cytometric immunophenotyping of LGLs from the peripheral blood and bone marrow samples revealed the same phenotype with expression of CD2, CD3, CD5 (dim), and CD8, but not CD4, CD16, or CD56. The CD7 expression was partial and dim. Serum was negative for hepatitis B surface antigen and antibodies for hepatitis C virus and human T cell lymphotropic virus (HTLV)-I/II. A clonality study using the BIOMED-2 protocols revealed clonal proliferation for T cell receptor (TCR) c chain gene rearrangement. Three weeks later, his WBC count was normal with 42 % LGLs C.-L. Lin Division of Hemato-Oncology, Department of Internal Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan