T-cell Receptor Gamma Chain Gene Research Articles

Histopathologic, phenotypic, and molecular criteria to discriminate low-grade intestinal T-cell lymphoma in cats from lymphoplasmacytic enteritis.

BackgroundDifferentiation of low‐grade intestinal T‐cell lymphoma (LGITL) from lymphoplasmacytic enteritis (LPE) in cats is a diagnostic challenge for pathologists.ObjectiveCharacterize histologic, immunohistochemical, and molecular features of LGITL and LPE.AnimalsForty‐four client‐owned cats, 22 diagnosed with LGITL and 22 with LPE.MethodsProspective, cohort study. Clinical suspicion of LGITL or LPE was based on persistent gastrointestinal signs, unresponsive to empirical treatments. All cats underwent a standardized diagnostic evaluation, including biopsy (preferentially full‐thickness), and were diagnosed with LGITL or LPE after review of clinical, laboratory, sonographic, histologic, immunohistochemical, and clonality results.ResultsA monomorphic lymphocytic population (22/22, 100%) and in‐depth mucosal infiltration (15/22, 68%) were hallmarks of LGITL. Epithelial patterns (nests and plaques) were significantly more frequent in LGITL (11/22, 50%) than in LPE (1/22, 5%) cases (P = .001). A CD3+ lymphocytic apical‐to‐basal gradient was observed in 9/22 (41%) of LGITL vs 1/22 (5%) of LPE cases (P = .004). Most LPE cases (17/18, 94%) featured marked fibrosis in the superficial part of the lamina propria. The Ki‐67 20%‐ and 30%‐thresholds discriminated between LGITL and LPE within both the epithelium (specificity >95%) and lamina propria (specificity >95%), respectively. All LGITL cases were CD3+ pSTAT3− and pSTAT5+. T‐cell receptor gamma chain gene rearrangements indicated monoclonality in 86% of LGITL cases. Surprisingly, 70% of LPE cases featured monoclonality (40%) or monoclonality on a polyclonal background (30%).Conclusions and Clinical ImportanceWe identified new histologic, immunohistochemical, and clonality criteria to distinguish LGITL from LPE.

Clonal T-Lymphocyte Populations in Healthy Donors

IntroductionT-cell clonality analysis based on T-cell receptor gamma chain (TCRG) genes rearrangements in peripheral blood could be useful for T-cell neoplasms diagnosis. Monoclonal products that exceed polyclonal background more than 3 times are often seen in lymphoid malignancies. Doubtful clonal results might be a possible feature of a relatively low tumor burden as well as a sign of a benign reactive process or reduction of the repertoire of lymphocytes associated with age. T-cell clonality in peripheral blood has been described in autoimmune diseases and viral infections. Examination of T-cell clonality in healthy people is necessary to define normal values of clonal variation and to develop algorithms for differential diagnosis of reactive and neoplastic conditions. Cytotoxic T-lymphocytes are the intended substrate of reactive processes. CD8 and CD57 are the characteristic surface markers of the cell population. Selection of CD8+ and CD57+ cells followed by determination of clonality based on TCRG genes rearrangements used to correlate clonal products with a specific functional population of lymphocytes and thereby to understand the basis of clonal expansion.Thus the aim of the research was to study the frequency of T-cell clonality and possible features of the clonal expansion of T-lymphocytes in healthy adults.Materials and methodsThe study included 25 healthy donors with a median age of 40 years (from 19 to 62 years). Samples of peripheral blood were obtained at the blood transfusion facility of the National Research Center for Hematology. All donors gave informed consent for participation in the research.CD8+ and CD57+ lymphocytes were isolated by means of immunomagnetic selection on Miltenyi Biotec beads. T-cell clonality was assessed by TCRG gene rearrangements using multiplex PCR with BIOMED-2 primer system and subsequent GeneScan analysis.ResultsBulk lymphocyte population in 2 of 25 donors (8%) was monoclonal, in 5 (20%) donors -doubtful monoclonal, in 2 donors (8%) - oligoclonal and in 16 of 25 donors (64%) - polyclonal (table 1). Thus there was the predominance of reactive clonal populations in the bulk population of cells in 28% (7/25) of donors.No correlation of the presence of clonal products with the age of donors has been demonstrated (Spearman test). According to the literature the frequency of clonal expansion of lymphocytes increases after 60 years. On the contrary, all participants in our study with the sole exception were younger. The median age of donors was 40 years. We suggest than at least in our donor sample T-cell clonality might be associated with individual characteristics of the donors rather than age related T-cell repertoire narrowing.Clonal products dominant in bulk population of lymphocytes, were attributed to CD8+ and CD57+ cells by means of magnetic selection. Therefore, the association of reactive clonal products with cytotoxic lymphocytes (figure 1, #8, #13) might be assumed.Additionally, in the case of donor #7 doubtful clonal product was discovered in the CD8- population, not previously dominant in the bulk population of lymphocytes and not identified in the CD8+ population (figure 1, #7).Since clear monoclonality of donor case #12 (figure 2) additional blood tests were run. No deviations in blood formula were found. Clinical evaluation confirmed the woman to be healthy, without any symptoms or history of chronic viral or autoimmune diseases.ConclusionsThe presence of clonal expansion of T-lymphocytes in peripheral blood should not be taken as a specific feature of lymphoid malignancy when seen in the absence of clinical or pathological signs of the disease. The typical cause of such conditions is the accumulation of clonal cytotoxic CD8+ CD57+ cells. The nature of clonal products in CD8- populations of lymphocytes are currently not clear and requires more extensive investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clonality testing in the lymph nodes from dogs with lymphadenomegaly due to Leishmania infantum infection.

IntroductionIn southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis.Materials and methods31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR.ResultsAll 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background.ConclusionInfections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.

Composite Peripheral T-Cell Lymphoma and B-Cell Lymphoma With Immunophenotype of Hairy Cell Leukemia: Report of an Unusual Case

Composite lymphoma (CL) is a rare form of lymphoma composed of two or more distinct types of malignant lymphomas involving a single anatomic location. CL of T and B cells is exceedingly rare. Hairy cell leukemia as part of a composite B- and T-cell lymphoma is rare. We present an unusual case of a CL of peripheral T-cell and B-cell lymphoma with immunophenotype of hairy cell leukemia. A 76-year-old woman presented with left cervical lymphadenopathy. PET scan was negative for other lymphadenopathy or organomegaly. She had a history of treated peripheral T-cell lymphoma in a different node 8 years back. She had neutropenia after chemotherapy. Bone marrow examination was normal. Excisional biopsy of the lymph node revealed architectural effacement by small lymphocytes intermixed with large atypical lymphoid cells demonstrating extensive nuclear irregularities and prominent nucleoli. Flow cytometry showed predominance of monoclonal B cells with expression of CD19, CD20, CD23, CD25, CD11c, and CD103, along with T cells expressing CD2, CD3, CD5, CD7, CD38, and CD4/CD8 ratio of 11:1. Immunohistochemistry revealed a predominant B-cell population with T cells expressing CD15 and CD30 and negative for ALK. PCR assays confirmed clonal rearrangements for T-cell receptor gamma and immunoglobulin heavy chain genes, establishing a diagnosis of composite B- and T-cell lymphoma. This was unusual as the B-cell immunophenotype was consistent with hairy cell leukemia, but the morphology and clinical presentation were not. Furthermore, coexpression of CD15 and CD30 in T-cell lymphoma was interestingly rare. CL of T and B cells poses a diagnostic challenge because one component may overshadow the other and the coexisting T cells are often marked as “reactive.” There are currently no agreed-on standards for treatment. This case highlights the importance of a systemic approach and ancillary techniques to arrive at this challenging diagnosis.

Role of high-throughput sequencing in the diagnosis of cutaneous T-cell lymphoma

AimsSubstantial clinicopathological overlap exists between cutaneous T-cell lymphoma (CTCL) and benign conditions, leading to diagnostic difficulties. We sought to delineate the utility of high-throughput sequencing (HTS) across a spectrum of...

An Unusual Posttransplant T-cell Lymphoma After Liver Transplantation: A Case Report

Posttransplantation lymphoproliferative disorders (PTLDs) encompass a spectrum of heterogeneous entities ranging from benign lymphocytic proliferations to high-grade malignant lymphomas. The majority of PTLDs are associated with reactivation of Epstein-Barr virus (EBV), which induces B-cell proliferation and occurs in the setting of severe immune suppression after solid organ or bone marrow transplantation. T-cell/natural killer cell PTLDs are relatively rare, constituting ∼15% of all cases. T-cell PTLDs are usually aggressive, and outcomes are poor. This article describes an unusual case of T-cell PTLD with a favorable outcome. The patient is a 57-year-old man who underwent a liver transplantation due to hepatitis C cirrhosis. He developed graft-versus-host disease with skin and gastrointestinal involvement and generalized lymphadenopathy 4 months after transplantation. Histologic sections of an excised axillary lymph node showed atypical medium and larger T-lymphocytes that were positive for CD3, CD5, CD43, and CD8 but were negative for B-cell antigens, CD56, and in situ hybridization for EBV-encoded RNA. Polymerase chain reaction analysis revealed monoclonal T-cell receptor gamma chain gene rearrangement. A diagnosis of high-grade T-cell PTLD was made. The patient was treated with 4 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone and is currently in remission, 4 years after therapy. The rapid presentation of an EBV-negative T-cell PTLD with a nonaggressive course and complete response to treatment is an unusual presentation of posttransplantation T-cell lymphoma, which is usually associated with a high mortality rate.

Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats

Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation.The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.

Re-sequencing and assembly of chicken T cell receptor gamma locus

The genomic organization of the animal T cell receptor (TCR) loci is characterized by different gene families with high homology, and it is quite difficult to obtain accurate gene sequences and arrangements of these gene families. In this study, we identified the location of chicken TCR gamma chain (TCRγ or TRG) genes by comparing those TRG gene sequences with the chicken reference genome, and the corresponding bacterial artificial chromosome (BAC) clone, CH261-174P24, was chosen for further high-throughput DNA re-sequencing and assembly. As a result, a draft genome assembly containing ten scaffolds was obtained, which almost covered the chicken TRG gene locus and the flanking regions. Subsequently, the internal structure of these scaffolds was confirmed by PCR amplification and Sanger sequencing. Our analysis corrected two errors in the sequence-one near a TRG variable gene and one close to a gap, respectively, and several errors in the TRG variable genes in the chicken reference genome. In conclusion, our work has partially corrected the erroneously assembled sequences of the TRG gene locus in the chicken reference genome and thus provides a new method for genome sequence analysis of chicken TRA/D and TRB gene loci.

Simultaneous Occurrence of Angioimmunoblastic T-cell Lymphoma and Plasma Cell Leukemia

A 73-yr-old man was evaluated for fever and multiple palpable nodules in his neck and inguinal areas. Biopsy of a cervical lymph node demonstrated polymorphic infiltrates including T-cells with abundant clear cytoplasm (clear cells), follicular dendritic cells, and plasma cells in the background of prominent arborizing high endothelial venules. The involved lymph node had typical histological and immunohistochemical properties characteristic of angioimmunoblastic T-cell lymphoma (AITL), except for the absence of CD10 expression. Lymphoplasmacytic lymphoma was excluded. A staging computed tomographic scan showed generalized lymphadenopathy in his chest, abdomen, and pelvis, as well as hepatosplenomegaly. Laboratory findings demonstrated the presence of leukocytosis (30.5×109/L). A peripheral blood (PB) smear revealed rouleaux formations and numerous circulating plasma cells, with plasmacytoid lymphocytes accounting for 25% of all nucleated cells. Protein electrophoresis and immunofixation studies of serum indicated IgMκ monoclonal gammopathy (IgM level, 53.8 g/L; normal range, 0.4-2.3 g/L). A bone marrow (BM) biopsy revealed 80% hypercellularity with focal clusters of CD3+ malignant T-cells and diffuse CD138+ clonal plasma cells at proportions of 10% and 30%, respectively (Fig. 1). The CD3+ T-cells were medium-sized lymphoid cells with moderate amounts of clear cytoplasm and slightly indented nuclei with coarse and condensed chromatin. In concordance with the monoclonal gammopathy found in the serum, immunohistochemical analysis of mixed plasma cells revealed a pattern restricted to κ light chain reactivity. Rare scattered cells were found positive for Epstein-Barr virus (EBV) by using in situ hybridization. Analysis for the expression of CD20 indicated relatively few mature B-cells. Fig. 1 Histologic findings of the bone marrow biopsy. (A) Bone marrow biopsy showed nodular lymphocytic infiltrates with malignant cells (Hematoxylin and Eosin, ×400). (B) The malignant cells were positive for CD3 (×400). (C, D) Plasma cells ... Flow cytometry analysis of circulating lymphocytes revealed a B-cell population expressing CD19, but the cells lost CD20 without surface Ig expression (25.12% of total events). Instead, these cells intensely expressed CD38 with a fraction of the cells also expressing CD138. Almost all CD38+ cells exhibited cytoplasmic κ restriction. In addition to the clonal plasma cells, T-cells that co-expressed CD3 and CD4 were also found to be positive for the known T-cell antigens CD2 and CD5 but negative for CD7. Consistent with the immunohistological findings from primary lymph node, T-cells were found to be negative for CD10. T-cells with immunophenotypic variation were considered AITL cells and comprised up to 0.25% of the total gated events. Representative scatter plots of the immunophenotypic findings obtained from the flow cytometry analysis are shown in Fig. 2. Fig. 2 Flow cytometry analysis of circulating lymphocytes. (A) and (B) CD45 and side scatter (SSC) gating: T-cells were positive for CD3, and B-cells were positive for CD19. (C) and (D) The CD19+ B-cells expressed high density of CD38 and cytoplasmic monoclonal ... PCR analysis of the T-cell receptor gamma chain gene (TCRγ) demonstrated a clonal rearrangement that confirms T-cell clonality. Molecular analyses for the TCRγ rearrangement was performed through PCR by using a mixture of consensus V and J region primers. The PCR products were analyzed by nonradioactive single-strand confirmation polymorphism. Karyotypic analysis of the BM cell culture using conventional G-banding showed 46,XY in all metaphase cells analyzed (n=20). Consequently, the patient was diagnosed as having BM involvement of AITL and plasma cell leukemia (IgMκ) simultaneously. Following initial diagnosis, the patient received the first cycle of chemotherapy consisting of vincristine, doxorubicin, cyclophosphamide, and prednisolone. The patient also suffered from respiratory failure combined with septic shock and was managed at the intensive care unit. Following discharge, the patient was followed up to monitor the disease in an outpatient clinic, without further chemotherapy. AITL, one subtype of peripheral T-cell lymphoma, is frequently associated with polyclonal B-cell or plasma cell proliferation [1]. The B-cell expansion observed in AITL has been proposed to be related to the functions of neoplastic follicular helper T-cells. However, proliferation of clonal B-cells or plasma cells has rarely been reported [2, 3, 4]. We hypothesized that the clonal plasma cell population is unrelated to the patient's AITL status. In over 20% of clonal plasma cells in PB and BM, the presence of M-protein in serum and absence of EBV could support this possibility. Factors contradicting plasma cell neoplasm include the followings: negative organ impairment including hypercalcemia; renal insufficiency; anemia; bone lesions; and IgM paraprotein (non-IgG or IgA) that could be associated with a clone of lymphoplasmacytic cells [5]. However, there was no evidence of CD20+ neoplastic B-cells in BM. In conclusion, the current case emphasizes that AITL could present with marked proliferation of clonal plasma cells, thereby potentially masking underlying T-cell lymphoma. Being aware of this possible effect is clinically important when determining treatment strategies and prognostic stratification, especially in the patients with a clonal B-cell population.

First Description Of a t(10;11)(q22;q23)/MLL-TET1 Reciprocal Translocation In a T Lymphoblastic Lymphoma With Subsequent Lineage Switch To Acute Myelomonocytic Myeloid Leukemia

TET1 genomic breakpoints and clinical features of MLL-TET1 rearrangements have been described in 13 acute leukemia cases, 11 in AML, 2 in B cell-precursor ALL. The incidence of this rare translocation was evaluated to 0.3% of MLL rearranged AML cases (5 out of 1590 MLL [Meyer C. Leukemia 2013]). Although those cases are very uncommon, their study can improve our current understanding of leukemogenesis. We report here the first t(10;11) MLL-TET1 positive case of lymphoblastic T lymphoma occurring in a 31 years old male patient, with a subsequent transformation to AML.The patient was referred for a large mediastinal mass and right pleural effusion. Mediastinal and bronchus biopsies led to the diagnosis of a precursor-T cell lymphoblastic lymphoma, expressing CD3, CD5, CD4, CD8, CD10 antigens, without any expression of CD34 or CD79.Molecular analyses of the malignant T-cells showed a clonal TCR gamma-chain gene rearrangement together with HOXA10 overexpression. FISH analysis showed a MLL breakage. The partner gene, TET1, was identified using RP11-9E13 and RP11-314J18 BACs, corresponding to the recurrent translocation t(10;11)(q22;q23). MLL-TET1 fusion transcript was detected (intron 8 of TET1 fused to exon 8 of MLL), as well as its reciprocal transcript.The patient was treated following the (GELA) LL03 protocol, and was considered in complete remission after induction and consolidation phases. Fourteen months after the diagnosis, a bone marrow examination was performed for thrombopenia (6 G/L) which revealed a myelomonocytic acute leukemia with trilineage dysplasia.At this time, The MLL-TET1 fusion transcript as well as HOXA10 overexpression was still present but the TCR rearrangement was not detected. A non-familial donor allogeneic bone marrow transplant was performed in CR after intensive chemotherapy, that was complicated by a grade IV acute graft-versus-host disease. The patient died 54 days after the transplant of bacterial sepsis leading to multi-organ failure.MLL-TET1 fusions have been described in 13 cases in the literature, mainly in AMLs (11/13 cases, mostly AML M4 or M5 cases) and in B-ALLs (2/13 cases). The case reported here presents two interesting features. Firstly, this patient harbors the first MLL-TET1 fusion reported to date in T-ALL, and secondly this case presented a lymphoid to myeloid phenotype switch during the time course of the disease.This strongly suggests that the translocation occurred very early during hematopoietic differentiation, prior to the lymphoid/myeloid commitment. As described for the Ph1 chromosome rearrangement, the t(10;11) translocation may arise in hematopoietic stem cell rather than in committed progenitors. These features are also described in 8p11 stem cell syndrome that involves FGFR1. In both cases, genetic rearrangements arise in myeloid or lymphoid neoplasms with possible subsequent transformation.TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and play a key role in active DNA demethylation. TET1 and TET2 are also the key enzymes responsible for the presence of 5hmC in mouse embryonic stem cells (ESCs) (Koh KP., Cell Stem Cell 2011), and TET1 functions to regulate the lineage differentiation potential of ESCs. In addition to its role in DNA demethylation, TET1 interacts physically with NANOG and NANOG/TET1 co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells (Costa Y., Nature 2013). Taken together, these observations enlighten possible mechanism of the lineage switch observed in this case, and may be a rationale for using demethylating agents in MLL-TET1 neoplasms. Disclosures:No relevant conflicts of interest to declare.

Neuropathy in a patient with lymphocytic thrombophilic arteritis

A 35-year-old Lebanese woman presented with a 3-year history of persistent, localized livedo racemosa over her feet, distal legs and forearms that was associated with the development of lower limb sensorimotor neuropathy. Investigations revealed the patient was heterozygous for prothrombin gene mutation and was also found to have a T-cell receptor gamma chain gene rearrangement. Histological examination revealed a mid-lower dermal medium vessel lymphocytic vasculitis with prominent fibrinoid ring within its wall. These findings are consistent with a recently described condition known as lymphocytic thrombophilic arteritis. This has so far been considered to be a benign clinical condition not associated with extra cutaneous manifestations. The novel findings in the present case are the associated sensorimotor neuropathy and the characteristic fibrin ring appears to be intramural rather intraluminal in location. The findings of a T cell gene rearrangement and a prothrombin gene mutation suggest that both immunological and thrombophilic factors might contribute to the development of this condition.

Follicular variant of peripheral T-cell lymphoma mimicking follicular lymphoma: A case report with a review of the Literature

Peripheral T-cell lymphoma (PTCL) with a follicular growth pattern is very rare. Herein, a case of follicular variant of PTCL in a 50-year-old man who complained of tonsillar and generalized lymph node swelling is reported. The resected tonsil revealed a vague nodular growth pattern of atypical cells, medium to large in size, with abundant pale cytoplasm. The lymphoma cells were CD3(+) CD4(+) CD5(+) CD8(-) CD10(+) CD56(-) CD57(-) BCL6(+) PD-1(+) CXCL13(+) and were associated with a meshwork of CD21(+) follicular dendritic cells. Molecular studies revealed clonal rearrangement of the T-cell receptor gamma chain gene but not of the immunoglobulin gene. Cytogenetic analysis disclosed a complex abnormality in 18 of 20 cells with the exclusion of t(5; 9). These findings suggest that the present case is a follicular variant of PTCL derived from follicular T-helper cells.

Cutaneous T-cell lymphoma occurring with a melanocytic proliferation, masquerading as a nonhealing ulcer with reactive changes

Two of the most challenging areas in dermatopathology are lymphoproliferative disorders and melanocytic lesions. We present a case of peripheral T-cell lymphoma occurring with an intradermal melanocytic proliferation. A 63-year-old Caucasian man presented with a 12-cm edematous, erythematous to violaceous, scalp ulceration that had enlarged over six months. Previous biopsies showed reactive changes which were concerning for infection. The last biopsies showed small to intermediate sized, angulated cells with clear cytoplasm within the dermis, with extension into the epidermis. These cells stained positive with markers for CD3, CD45RO and CD43, yet showed decreased expression of pan-T-cell markers CD5 and CD7, and absent expression of CD4, CD8, CD56 and CD57 and EBV. Molecular studies showed a clonal T-cell receptor gamma chain gene rearrangement. The diagnosis was peripheral T-cell lymphoma, unspecified. Another biopsy from an indurated area separate from the ulcer showed scattered, enlarged cells embedded in the same lymphocytic infiltrate. No mitotic figures were identified. These cells stained for S100 and Melan-A, in a partly nested arrangement. This was felt to represent a melanocytic nevus. This case likely represents an extraordinary coincidence of two distinctly different neoplasms.

Hemophagocytic lymphohistiocytosis associated with myelodysplastic syndromes

Hemophagocytic lymphohistiocytosis/hemophagocytic syndrome (HLH/HPS) is a reactive disorder of the mononuclear phagocytic system, characterized by generalized histiocytic proliferation with marked hemophagocytosis in the bone marrow [1]. It comprises two different categories: a primary (genetic) and a secondary (acquired) form. Malignant neoplasm-associated HPS (MAHS) is categorized as a secondary HLH. MAHS is mainly associated with lymphoma and rarely with other carcinomas [2]. To our knowledge, HLH associated with myelodysplastic syndromes (MDS) has not been described, although MDS with hemophagocytosis has been observed. Here, we describe a case of HLH associated with MDS, which presented with abundant CD8 T cells in the bone marrow and elevated plasma-soluble interleukin 2 receptor (sIL-2R). A 60-year-old Japanese man was admitted with pancytopenia, epitaxis, fever and general fatigue. A hematological examination showed a hemoglobin concentration of 6.5 g/dL, a platelet count of 14,000/lL and a leukocyte count of 3,200/lL with 42.8% atypical lymphocytes (Fig. 1a). Blood biochemistry showed increased levels of lactate dehydrogenase (LDH) (331 IU/L), ferritin (649 ng/mL) and sIL-2R (4,054 U/mL). NK cytolytic activity was measured by the standard 51-chromium (Cr) release assay. NK cytolytic activity was not reduced at both the E/T ratios of 10:1 (13.1%, normal range 8.9–29.5%) and 20:1 (22.5%, normal range 17.1–48.7%). Serological studies for hepatitis B, hepatitis C and HTLV-1 were negative. IgM antibodies to parvovirus were negative. Serological test for EBV revealed that anti-viral capsid antigen IgM was negative in the presence of anti-viral capsid antigen IgG (1609) and anti-EBV nuclear antigen antibody (409). The number of EBV DNA copies in serum was under 2.0 9 10 copies/mL (normal \200 copies/mL). Computed tomography (CT) revealed hepato-splenomegaly, ascites and bilateral pleural effusion. Bone marrow aspirate showed dyserythropoietic changes (abnormally lobulated nuclei) (Fig. 1c) and dysplastic megakaryocytes (micromegakaryocytes) (Fig. 1d). Erythrocytic precursors were 10.4% and bone marrow myeloblasts were 2.0% (Fig. 1e). Chromosome analysis showed severe complex karyotype: 54–57XY, ?1[2],?3[2],?4[4],?6[4],?8[4],add(9)(p22)[2],?11[3],add (15)(p11.2)[4],add(16)(q24)[4],add(19)(p13.1)[4],add(20) (p13)[4],?21[2],2–5mar[cp4]/46,XY[8]. A diagnosis of refractory anemia with excess blasts-1 (WHO classification) was made. In addition, bone marrow examination revealed increased numbers of atypical lymphocytes (Fig. 1b) and multiple sites of active hemophagocytosis (Fig. 2a–c). Atypical lymphocytes in bone marrow were positive for CD2, CD3 (surface, cytoplasmic), CD5, CD7, CD8, HLA-DR, TCR-ab, and negative for CD4. No monoclonal rearrangement of TCR beta, gamma and delta chain genes was detected by Southern blotting. These findings suggested that the atypical lymphocytes were reactive CD8 T cells. Combining fever, hemophagocytosis, splenomegaly, increased levels of ferritin and sIL-2R, a diagnosis of HLH was established. Although steroid therapy was administered for HLH, pancytopenia was not improved. Two months after the initial diagnosis, the levels of LDH and ferritin were further elevated (LDH 1,474 U/mL, ferritin 4,347 ng/mL). Bone marrow examination T. Tsuji (&) H. Yamasaki H. Tsuda Division of Hematology and Oncology, Kumamoto City Hospital, Kotoh 1-1-60, Kumamoto 862-8505, Japan e-mail: tsuji.takahiro@city.kumamoto.lg.jp

Posttransplantation lymphoproliferative disease involving the pituitary gland

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.

Monitoring of Minimal Residual Disease (MRD) after Multidrug Chemotherapy and Its Correlation to Outcome in Dogs with Lymphoma: A Proof-of-Concept Pilot Study

Tumor cell burden in dogs with lymphoma cannot be assessed accurately by diagnostic evaluation during clinical complete remission (CR). Recent advances in polymerase chain reaction (PCR)-based methods enabled us to quantify minimal residual disease (MRD) in canine lymphoma. To quantify MRD in dogs with lymphoma treated with multidrug chemotherapy and to correlate it with remission duration after chemotherapy. Seventeen dogs with lymphoma that achieved CR by multidrug chemotherapy. Rearranged immunoglobulin heavy chain or T-cell receptor gamma chain gene fragments from lymphoma cells were PCR amplified and sequenced to prepare clone-specific primers and probes for real-time PCR to quantify MRD. MRD in the peripheral blood was monitored during and at the end of a 25-week multidrug chemotherapy protocol. Correlation between MRD at the end of chemotherapy and remission duration after chemotherapy was analyzed. MRD gradually decreased after initiation of multidrug chemotherapy, reached a nadir as low as <0.019-1.0 cells/microL at weeks 4-17, and remained low or slightly increased until week 25. MRD at the end of chemotherapy was negatively correlated with remission duration from the end of chemotherapy to relapse. MRD could be an objective marker to indicate tumor cell burden in dogs with lymphoma even in clinical CR. MRD at the end of chemotherapy could be a prognostic factor to predict remission duration after chemotherapy.

Primary cutaneous T-cell lymphoproliferative disorder of donor origin after allogeneic haematopoietic stem-cell transplantation

A 56-year-old male patient had a history of mantle-cell lymphoma, which was treated with polychemotherapy and reduced-intensity conditioning allogeneic haematopoietic stem cell transplantation (ASCT) from his healthy sister with an identical human leucocyte antigen profile. Six years after transplantation, the patient developed asymptomatic eczema-like cutaneous lesions. Histologically the lesions contained a dense superficial lichenoid infiltrate, mainly consisting of CD4+ atypical medium to large lymphocytes showing indented hyperchromatic nuclei. In situ hybridization for Epstein-Barr virus was negative. PCR amplification of the T-cell receptor-gamma chain gene from several lesions revealed a monoclonal rearrangement without clonal variation. Two-colour fluorescence in situ hybridization (X and Y chromosomes) and microsatellite genotyping were used to compare samples from the patient (transplant recipient), his sister (donor) and the skin biopsy sample, which confirmed that the origin of the neoplastic cells was the donor graft. To our knowledge, this is the first case of post-transplant primary cutaneous T-cell lymphoproliferative disorder after ASCT.

Composite gastrointestinal lymphoma consisting of diffuse large B-cell lymphoma and peripheral T-cell lymphoma

Composite lymphomas are defined as two different types of lymphomas presenting in the same anatomic site [1]. A rare case of a composite gastrointestinal lymphoma consisting of diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) is presented. A 65-year-old woman was diagnosed with mucosaassociated lymphoid tissue lymphoma of the duodenum in 2001. Eradication therapy for Helicobacter pylori resulted in complete remission of the lymphoma. After 4 years, she was admitted to our hospital due to hematemesis in December 2005. The levels of hemoglobin and soluble interleukin-2 receptor were 10.4 g/dL and 938.1 U/mL, respectively. Endoscopic examination revealed multiple ulcers in the stomach and ileum. On microscopy, both gastric and ileal ulcers showed diffuse proliferation of large lymphoid cells, which were positive for CD20, CD79a, and Epstein–Barr virus (EBV) encoded RNA-1 (EBER-1) in situ hybridization (ISH) and negative for CD3 and CD45RO (Fig. 1a–d). These findings were compatible with EBV-positive DLBCL of the elderly, according to the new WHO classification [2]. After 3 courses of combination chemotherapy (pirarubicin, vincristine, cyclophosphamide and prednisolone; THP-COP) with rituximab, the ileal ulcers disappeared, but the area of the gastric lymphoma enlarged. A partial gastrectomy was performed with a diagnosis of perforation of the stomach in April 2006. Unexpectedly, pathological examination of the resected specimen demonstrated diffuse proliferation of smallto large-sized lymphocytes with T-cell markers (positive for CD3, CD4, CD45RO, CD56 and TIA-1, and negative for CD8, CD20, CD79a and EBER-ISH, Fig. 1e–h). The diagnosis of PTCL was made, and the distribution of the disease and strong positivity of TIA-1 suggested enteropathyassociated T-cell lymphoma. Since the size of the lymphoma cells was mainly small to medium and they were positive for CD56, we speculated that the lymphoma could be classified into enteropathy-associated T-cell lymphoma type 2 [3, 4]. However, the mucosa of the specimen was almost destroyed and the distribution pattern was not clear. Retrospectively, we performed PCR analysis of T-cell receptor (TCR) gamma chain gene in the gastric specimen at the DLBCL stage in 2005 and PTCL stage in 2006 (Fig. 2). Although scattered T cells were present in the specimen, rearrangement of TCR was not detected. On the other hand, strong rearranged band was detected at the PTCL stage. Since PTCL was diagnosed during the course of chemotherapy against DLBCL and residual B-cell disease could not be denied after gastrectomy, the patient was given 2 courses of consolidation chemotherapy (etoposide, methylpredonisolone, cytarabine and cisplatin; ESHAP) with rituximab. Achievement of complete remission was confirmed by 18[F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan in June 2006. However, Y. Tachikawa M. Shiratsuchi E. Sada K. Idutsu J. Kiyasu R. Takayanagi Y. Abe (&) Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan e-mail: abey@intmed3.med.kyushu-u.ac.jp

Cutaneous Peripheral T-Cell Lymphoma Associated With a Proliferation of B Cells

Although the new World Health Organization-European Organization for Research and Treatment of Cancer classification focuses on providing uniformity in the diagnosis of cutaneous lymphomas, cutaneous peripheral T-cell lymphoma (PTL) remains a poorly defined subgroup. As follow-up to a study of systemic PTL complicated by a proliferation of B cells, we studied 16 cases of cutaneous PTL that contained morphologically atypical T cells associated with a significant infiltrate of B cells (about 20%-50%). A clonal T-cell receptor gamma chain gene rearrangement was present in all cases. In contrast, a clonal immunoglobulin heavy chain gene rearrangement was present in only 1 case. Clinical staging in 14 cases identified systemic involvement in 2. At last follow-up, both patients with systemic involvement had died of disease, and the majority of patients with primary cutaneous disease were alive (11/12). The presence of numerous atypical B cells and T cells caused diagnostic confusion in these cases. Comprehensive pathologic studies, coupled with clinical staging, are necessary for the accurate diagnosis of this unusual manifestation of cutaneous PTL.