Clinicopathologic features of adult EBV-associated B-cell lymphoproliferative disease.

In the present study, 21 cases of adult/late-onset EBV-associated lymphoproliferative disease (AELPD) with an uncertain malignant potential were investigated with regard to their histomorphology, immunophenotype, clonal rearrangement of the heavy chain (IgH) and T-cell receptor (TCR) genes and clinical course. The cases were histomorphologically reevaluated and assigned to one of three morphological groups: mononucleosis-like, Hodgkin-like, or polymorphous. In addition, cases with or without detectable necrosis were investigated for differences in clinical outcome. Overall survival was highest in the group with Hodgkin-like morphology (4/4 patients), followed by patients with mononucleosis-like phenotype (4/5 patients surviving). Cases with polymorphous morphology showed the poorest survival rates with 7/12 patients dead of disease (58%). 4/6 patients with histologically detectable necrosis died (66%), but only 4/15 patients without necrosis (27%). 11/21 cases with AELPD showed clonal rearrangement for IgH (n = 4), TCR (n = 5) or IgH + TCR (n = 2). 5/11 patients with clonal rearrangement died (45%), and this percentage was similar in all of the three subgroups. In conclusion, the present study shows that polymorphous morphology and detection of necrosis in AELPD are frequently linked to a fatal clinical course, whereas Hodgkin-like morphology seems to be associated with a more favourable prognosis. Clonal rearrangement of IgH or TCR is frequent in AELPD, but prognosis is unpredictable from this feature.

Detection of circulating tumor cells using GeneScan analysis for antigen receptor gene rearrangements in canine lymphoma patients.

The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients.

Optimal strategies to identify aberrant intra-epithelial lymphocytes in refractory coeliac disease.

INTRODUCTION: Different strategies have been developed to identify those refractory celiac disease (RCD) patients who are at risk to develop an enteropathy associated T-cell lymphoma (EATL). Flow cytometric analysis of intra-epithelial lymphocytes (IEL) with an aberrant phenotype is considered the golden standard but is not widely available. Immunohistochemistry (IHC) and T-cell receptor (TCR) rearrangement studies are commonly available but may lack sensitivity and specificity. Here, we compared the three different methods in the workup of patients suspected for RCD. METHODS: Duodenal biopsies from control patient (n = 5), RCD patients with moderately increased aberrant IEL populations (20-50 %: n = 14), and RCD patients with high numbers of aberrant IEL (>50 %: n = 5) as determined by flow cytometry were analysed by IHC and TCR-γ chain rearrangement analysis. Three pathologists scored the slides independently. RESULTS: Sensitivity of IHC and TCR-γ rearrangement analysis in RCD patients with high numbers of aberrant IELs was 100 and 71 %, respectively. RCD patients with aberrant cells between 25 and 50 % however, were missed by IHC and TCR in 50 and 57 % of cases, respectively. In addition, inter-rater reliability analysis of the IHC scoring revealed coder-pair Kappa coefficients between 0.28 and 0.85. CONCLUSION: Immunohistochemistry and to a lesser extent TCR-γ clonality analysis are sensitive in identifying patients with high numbers of aberrant IEL populations, yet miss half of RCD patients with moderately increased numbers. In addition, IHC has a high inter-observer variability. Therefore, patients suspected for RCD should undergo flow cytometric analysis of the duodenum.

A comparison of deep sequencing of TCRG rearrangements vs traditional capillary electrophoresis for assessment of clonality in T-Cell lymphoproliferative disorders.

OBJECTIVES: To design and evaluate a next-generation sequencing (NGS)-based method for T-cell receptor γ (TCRG) gene-based T-cell clonality testing on the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA) platform. METHODS: We analyzed a series of peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue specimens with NGS vs traditional capillary electrophoresis methods. RESULTS: Using a custom analysis algorithm that we developed, our NGS assay identified between 2,215 and 48,222 unique TCRG rearrangements in a series of 48 samples. We established criteria for assigning clonality based on parameters derived from both the relative and absolute frequencies of reads. In a comparison with standard capillary electrophoresis, 19 of 19 polyclonal samples and 24 of 27 samples that appeared clonal were in agreement. The three discrepant samples demonstrated some of the pitfalls of amplicon length-based testing. Dilution studies with T-lymphoid cell lines demonstrated that a known clonal sequence could be routinely identified when present in as few as 0.1% of total cells demonstrating suitability in residual disease testing. A series of samples was also analyzed on a second NGS platform and yielded very similar results with respect to the frequency and sequence of the clonal rearrangement. CONCLUSIONS: In this proof-of-concept study, we describe an NGS-based T-cell clonality assay that is suitable for routine clinical testing either alone or as an adjunct to traditional methods.

Distinct morphologic, phenotypic, and clinical-course characteristics of indolent peripheral T-cell lymphoma.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) consists of a heterogeneous group of lymphomas. Patients generally show an aggressive clinical course and very poor outcome. Although the 2008 World Health Organization classification of PTCL-NOS includes 3 variants, low-grade lymphoma is not included. Of 277 PTCL-NOS cases recorded in our consultation files, we examined the clinicopathologic characteristics of 10 patients with T-cell lymphomas composed of small-sized cells with slight nuclear atypia. Eight patients showed extranodal involvement (5 patients, spleen; 3 patients, thyroid), and 5 patients were at clinical stage I or II. Histologically, all samples presented diffuse infiltrate of small lymphoid cells, with few mitotic figures. Immunohistologically, all samples were positive for CD3, and CD20 was detected in 5 samples. All samples showed a low Ki-67 labeling index (mean, 1.05%), and 7 samples were positive for central memory T-cell markers. Clonal T-cell receptor γ chain and/or α-ß chain gene rearrangements were detected in all 10 patients. Five patients received chemotherapy, whereas for 3 patients, treatment consisted only of observation following surgical resection of the spleen or thyroid. Nine patients were alive at a median follow-up time of 19.5 months, whereas 1 patient died of an unrelated disease. The present study strongly indicates that T-cell lymphoma with small-sized lymphoma cells and a low Ki-67 labeling index is a distinct variant. Recognition of this novel lymphoma subtype, which should not be defined merely as PTCL-NOS, should be seriously considered.

Epstein-Barr virus-associated primary central nervous system cytotoxic T-cell lymphoma.

Primary central nervous system lymphoma (PCNSL) expressing T-cell markers is rare, among which nasal-type extranodal NK/T-cell lymphoma is an extremely rare subtype associated with Epstein-Barr virus (EBV) infection. Here we report the clinicopathologic features of a case of EBV-associated PCNSL showing a cytotoxic T-cell phenotype. The patient, a 73-year-old woman, presented with rapidly progressive mental deterioration. Brain MRI revealed multiple lesions with swelling in the bilateral cerebral hemispheres, which were hypointense on T1-weighted images, hyperintense on T2-weighted and fluid-attenuated inversion recovery images, and slightly hyperintense on diffusion-weighted images. Biopsy specimens from the temporal region showed many medium-sized anaplastic lymphocytic cells with perivascular and angio-invasive patterns in the cortex. Immunohistochemically, the cells were positive for CD3, CD8, T-cell-restricted intracellular antigen-1 (TIA-1), granzyme B and perforin, but negative for CD56 and CD20. In situ hybridization revealed EBV-encoded RNAs in the tumor cell nuclei. A rearrangement study showed T-cell receptor γ-chain gene rearrangement with a clonal appearance. The patient died 6 months after surgery, and a general autopsy revealed no lymphoma cells outside the brain. These cellular profiles are inconsistent with those of extranodal NK/T-cell lymphoma, and have not been previously described. This case appears to represent an unusual CNS manifestation of EBV-associated T-cell lymphoma.

Follicular peripheral T-cell lymphoma expands the spectrum of classical Hodgkin lymphoma mimics.

Epstein-Barr virus (EBV)-infected B cells with Reed-Sternberg-like cell (RS) features may occur in peripheral T-cell lymphomas (PTCLs), especially in angioimmunoblastic T-cell lymphoma. Here, we report 5 patients presenting with lymphadenopathy whose first biopsies demonstrated nodular lymphoid proliferations containing scattered CD30+, CD15+, EBV+ Hodgkin and Reed-Sternberg-like cells, which led to an initial diagnosis of lymphocyte-rich classical Hodgkin lymphoma. However, the uncommon clinical features and/or the occurrence of relapse as PTCL prompted review of the biopsies with expanded immunohistologic and molecular studies and revision of the diagnoses to follicular variant of PTCL (F-PTCL). All cases had atypical small to medium-sized CD3+ T cells that expressed CD10 (4/5) and the follicular helper T-cell (TFH) antigens BCL6, PD1, CXCL13, and ICOS. All demonstrated clonal T cells with a similar pattern in multiple samples from 4 patients. In 2 cases, flow cytometry demonstrated circulating lymphocytes with an abnormal sCD3+, CD4+, ICOS+ immunophenotype. Two patients had a skin rash at presentation, and 1 had B symptoms. Two of the 4 patients treated with polychemotherapy are alive at 3 and 6 years after first diagnosis. These cases highlight how some F-PTCLs may closely mimic lymphocyte-rich classical Hodgkin lymphoma requiring careful assessment of the T cells before rendering the latter diagnosis. The functional properties of TFH cells might lead to the presence of EBV-positive B blasts with RS-like features in TFH-derived PTCL such as angioimmunoblastic T-cell lymphoma and F-PTCL.

Manual microdissection technique in a case of subcutaneous panniculitis-like T-cell lymphoma: a case report and review.

Demonstration of T-cell receptor gene monoclonality often plays an important role in the diagnosis of T-cell lymphoma. When a test to detect monoclonality is performed on whole tissue sections, the presence of a reactive lymphocyte population may reduce sensitivity. This may be especially true for early or borderline cases of lymphoma. Microdissection techniques may be utilized to more readily identify a clonal population of lymphocytes. Subcutaneous panniculitis-like T-cell lymphoma represents a cutaneous lymphoid neoplasm whose clinical course may vary from an indolent, waxing and waning course to an aggressive course resulting in death. We report the first case of a microdissection technique used to facilitate diagnosing a case of subcutaneous panniculitis-like T-cell lympoma.

TCRß clonality improves diagnostic yield of TCRγ clonality in refractory celiac disease.

BACKGROUND: Refractory celiac disease (RCD) is a preneoplastic condition as many patients develop an enteropathy-type T-cell lymphoma, a mature T-cell receptor α-ß lymphoma arising in the gut with an ominous outcome. Recently, research focused on a population of intraepithelial intestinal lymphocytes expressing the same lymphoma T-cell receptor variable region (V)γ, as shown by polymerase chain reaction (PCR) analysis and sequencing. Meanwhile, the Biomedicine and Health-2 Concerted Action has made available standardized, highly specific, and sensitive PCR assays not only for Vγ but also for Vß. GOALS: We verified whether analyzing both rearrangements in duodenal biopsies from RCD patients increases the diagnostic accuracy of this method. STUDY: Duodenal biopsies were analyzed from 15 RCD patients, 21 negative controls, and 2 positive controls (enteropathy-type T-cell lymphoma complicating celiac disease). Multiplex clonality analyses were performed according to the Biomedicine and Health-2 protocols. PCR products were cloned and sequenced. RESULTS: Monoclonal rearrangements were found in 5/15 samples from patients with RCD (both rearrangements in 2 cases, Vß only in 2, and only 1 solitary Vγ clonality). Monoclonality was found in 4/8 of the RCD patients who subsequently died, whereas only 1/7 of the patients still alive presented a monoclonal rearrangement. Positive controls revealed both monoclonal rearrangements; rearrangements were not detected in 20 of 21 negative controls. Sequencing of the amplified fragments confirmed the results. CONCLUSIONS: The combined analysis of both rearrangements allowed recognition of monoclonal populations in otherwise negative patients, with detection rates from 20% (Vγ only) to 33% (Vγ and Vß), thus raising the likelihood of early identification of RCD patients at high risk of death.

The significance of multiplex PCR/heteroduplex analysis-based TCR-γ gene rearrangement combined with laser-capture microdissection in the diagnosis of early mycosis fungoides.

BACKGROUND: The diagnosis of early mycosis fungoides (MF) is a big challenge to dermatologists and dermatopathologists because it lacks specific clinicopathologic features. METHODS: Fifty-two paraffin-embedded skin samples from 50 patients, including 31 with suspected MF, 10 with typical MF and 9 with benign inflammatory dermatosis (BID), were obtained from our archives. DNA was extracted both by traditional phenol-chloroform method and by the laser-capture microdissection (LCM)-proteinase K approach. The T(VG) /T(JG) , V(2-5) /V(8-12) /JGT(1) and BIOMED-2-TCR-γ primers were used to assess TCR-γ monoclonal rearrangement as measured by polymerase chain reaction (PCR). RESULTS: In the suspected MF group, clonal TCR-γ gene rearrangements were detected in 11/31 cases (35.5%) by phenol-chloroform DNA extraction and in 25/31 cases (80.7%) by LCM-proteinase K extraction (p < 0.05). While T-cell clonality was detected in 8/10 cases (80%) by the phenol-chloroform method and 10/10 cases (100%) by LCM (p > 0.05) in the typical MF group, no TCR-γ monoclonal rearrangement was detected in the BID group. CONCLUSIONS: The strategy of multiple PCR/heteroduplex analysis for TCR-γ gene rearrangement combined with LCM increases the detection rate of clonal TCR-γ gene rearrangement in early MF cases and could provide strong evidence to confirm the diagnosis of early MF.

GeneScan analysis to detect clonality of T-cell receptor γ gene rearrangement in feline lymphoid neoplasms.

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity.

Rearrangement patterns of the canine TCRγ locus in a distinct group of T cell lymphomas.

The T cell antigen receptor chains are assembled through a rearrangement process that combines variable (V), diversity (D) and joining (J) region genes. Recently, the entire canine T cell receptor γ (TRG) locus was described. It is arranged in 8 cassettes with up to 3 V genes, 2 J genes and 1 C gene each. However, no data is available beyond the level of sequence analysis. The objective of this study was to identify rearranged genes of the canine TRG locus through experimental analysis and to assess gene usage and patterns of rearrangement in a series of canine T cell lymphomas. Rearranged genes were identified through computational analysis of recombination signal sequences (RSSs), a gene's potential to generate a polyclonal smear, and through sequencing of clonal rearrangements in a series of T cell lymphomas. Out of a total of 32 Vγ and Jγ genes, 21 genes were found to rearrange, 8 genes were considered not rearranged and 3 genes were suspected to rearrange but their status could not be determined definitely. Rearrangements of the canine TRG locus were assessed in a group of canine T cell lymphomas as well as 3 neoplastic T cell lines. An average of 4.6 rearrangements per lymphoma was found suggesting that canine T cells routinely rearrange multiple cassettes per allele. The most commonly rearranged Vγ genes belonged to subgroups Vγ2, Vγ3, and Vγ7. Genes in cassettes 2 and 3 preferentially rearranged within their respective cassettes, while Vγ genes in cassette 7 rearranged to a Jγ gene in cassette 8. There was a strong preference for Vγ2 genes to rearrange to a 3' Jγ gene and for Vγ3 and Vγ7 genes to rearrange to a 5' Jγ gene. This rearrangement pattern coincided with the conservation of the spacer sequence between V and J gene subgroups rather than the topologic location of genes. These data show that highly divergent spacer sequences allow for equally efficient recombination and suggest that spacer sequences can mediate compatibility between V and J genes.

Traumatic ulcerative granuloma with stromal eosinophilia: report of a case and literature review.

Traumatic eosinophilic granuloma with stromal eosinophilia is a rare entity that affects the oral mucosa and has a controversial etiologic pathogenesis. Histologically, these lesions are characterized by a dense and deeply infiltrative lymphoproliferation showing epitheliotropic characteristics and massive eosinophilia. Frequently, a population of mitotically active, atypical mononuclear cells can be noted. This report describes a case of traumatic eosinophilic granuloma with stromal eosinophilia in the floor of the mouth of an 88-year-old man. The phenotypic and genotypic profiles of the inflammatory infiltrate and large atypical mononuclear cells, using immunohistochemical and polymerase chain reaction-based molecular analysis, were analyzed.

Clonality in sarcoidosis, granuloma annulare, and granulomatous mycosis fungoides.

The histological discrimination of granulomatous cutaneous T-cell lymphomas (CTCLs) from reactive granulomatous disorders such as sarcoidosis and granuloma annulare (GA) may be difficult due to overlapping histological features. We analyzed the T-cell receptor gene rearrangement in sarcoidosis and GA to investigate the value of the detection of clonal T cells as an adjunctive diagnostic marker in the differentiation between sarcoidosis and GA versus granulomatous CTCLs. Rearrangement of T-cell receptor γ genes was examined by the use of automated high-resolution polymerase chain reaction fragment analysis in 35 cases of sarcoidosis and 15 cases of GA and compared with a series of 19 cases of granulomatous CTCLs. A monoclonal T-cell population was found in none of the cases of sarcoidosis and in 2 of 15 cases of GA (13%). In granulomatous CTCLs, a neoplastic T-cell clone was detected in 94%. Presence of clonal T cells argues in favour of a granulomatous CTCL, while a polyclonal T-cell population makes the presence of a sarcoidosis or a GA more likely. The analysis of T-cell clonality is a useful diagnostic adjunct in the differentiation between sarcoidosis and GA from granulomatous CTCLs.

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.

CD20-positive T-cell lymphoma with indolent clinical behaviour.

T-cell lymphomas that are positive for CD20 are very rare and most reported cases have demonstrated an aggressive clinical course. An unusual case of a 57-year old female who presented with recurrent enlarged lymph nodes for 12 years is reported. The lymph nodes from both 1995 and 2007 showed effacement of the lymph node architecture by a diffuse and dense infiltrate of small lymphoid cells. In terms of T- and B-cell markers, these small lymphoid cells were immunohistochemically positive for CD2, CD3, CD5, CD43, CD45RO and CD20, and were negative for PAX5, CD79a and cyclin D1. Molecular genetic analysis showed T-cell receptor-gamma chain gene rearrangement. Recognition of this type of CD20-positive T-cell lymphoma is important for ensuring a correct diagnosis so that the patient can be offered the most appropriate therapy. The indolent behaviour of the present case is unusual and awaits further clinical follow-up and laboratory investigation.