Subcutaneous panniculitis-like T-cell lymphoma: Morphological, immunophenotypical and clonality assessment in six cats.

BACKGROUND: Primary cutaneous lymphoma represents 0.2%-3% of all feline lymphomas, with nonepitheliotropic lymphomas being the most common. In humans and dogs, subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a primary nonepitheliotropic lymphoma with a T-cell phenotype developing in the subcutis and often mimicking inflammation. OBJECTIVE: The aim of this report is to describe pathological, phenotypical and clonal features of SPTCL in cats. ANIMALS: Six cats with SPTCL were included in this study. MATERIALS AND METHODS: Skin biopsies were formalin-fixed, routinely processed and stained. Histological and immunohistochemical investigation for anti-CD18, CD204, CD79a, CD20, CD3, FeLVp27and FeLVgp70 and clonality assessment were performed. RESULTS: Four male and two female domestic shorthair cats, mean age 11.2 years, developed SPTCL in the abdominal (three), inguinal (two) and thoracic (one) regions. Variably pleomorphic neoplastic lymphoid cells were present in the panniculus in percentages, expanding the septa (six of six) and extending into fat lobules in one of six cats. Tumours were associated with elevated numbers of neutrophils (five of six), lesser macrophages (six of six) and variable necrosis (six of six). Neoplastic cells expressed CD3+ (six of six), with clonal T-cell receptor rearrangement detected in five of six cats. CONCLUSIONS AND CLINICAL RELEVANCE: This is the first description of SPTCL in cats. Lesions can be confused with panniculitis, leading to delay in diagnosis and therapy. Awareness of this neoplastic disease is relevant to avoid misdiagnoses and to gain greater knowledge about the disease in cats.

Possible Concomitant Aggressive NK Cell Leukemia and EBV-positive T-cell lymphoma; Using the online beta version of WHO-HAEM5 and videoconferencing software to make diagnoses accessible in an emerging economy.

BACKGROUND: Using the World Health Organization Classification 5th edition (beta version online; WHO-HAEM5bv) in emerging economies is key to global healthcare equity. Although there may be ongoing updates, hesitancy in accepting and reporting these diagnoses in publication conflicts with the WHO's commitment to global accessibility. Aggressive NK cell leukemia (ANKL) and systemic EBV-positive T-cell lymphoma of childhood (SEBVTCL) with CD4-positive immunophenotype are both rare entities, are most described in Asians and East Asians, are associated with prior systemic chronic active EBV disease (CAEBV), and presentation with Hemophagocytic Lymphohistiocytosis (HLH). Recognizing and diagnosing any one of these entities requires not only training and experience in hematopathology, but good cooperation between clinical physicians and all areas of the laboratory. We describe a 30-year-old woman who presented to a Vietnam hospital and was rapidly diagnosed with ANKL, SEBVTCL, and HLH using WHO-HAEM5bv essential criteria, aided by expert consultation from a United States (US) board certified hematopathologist in real-time using video conferencing software. METHODS: Zoom™ videoconferencing software; Immunohistochemistry; flow cytometric immunophenotyping; polymerase chain reaction (PCR), Next Generation Sequencing (NGS). RESULTS: At the time of hospital admission, automated complete blood count (CBC) with differential count showed slight anemia, slight lymphocytosis, and moderate thrombocytopenia. HIV serology was negative. Whole blood PCR for EBV was positive showing 98,000 copies/ml. A lymph node biopsy revealed histology and immunohistochemistry consistent with the online beta version WHO-HAEM5 classification of SEBVTCL arising in CAEBV. Blood and bone marrow studies performed for staging revealed no histologic or immunohistochemical evidence of T-cell lymphoma in the bone marrow core, however, atypical blood smear lymphocyte morphology and blood immunophenotyping by flow cytometry were consistent with WHO-HAEM5 classification of ANKL. NGS revealed no evidence of genetic variant(s) associated with HLH in Vietnam. All laboratory studies were performed at Blood Transfusion Hematology Hospital (BTHH) in Ho Chi Minh City Vietnam. CONCLUSION: Although Vietnam, an emerging economy, currently lacks the laboratory infrastructure to more rigorously confirm a rare synchronous presentation of two distinct EBV-driven T/NK cell neoplasms, these two concomitant diagnoses were made using only laboratory techniques available in Vietnam with the help of WHO-HAEM5bv and real-time video consultation by a US hematopathologist.

Molecular diagnosis of T-cell lymphoma: a correlative study of PCR-based T-cell clonality assessment and targeted NGS.

Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.

BACKGROUND: Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vß repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant ß chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. METHODS: Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. RESULTS: We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. CONCLUSIONS: Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

RHOA G17V mutation in angioimmunoblastic T-cell lymphoma: A potential biomarker for cytological assessment.

BACKGROUND: The World Health Organization, in a 2016 revision, introduced recurrent genetic abnormalities for classifying mature T- and NK-cell neoplasms. However, the role of genetic analyses from lymph node aspiration cytology is still not elucidated. We hypothesize that the use of genetic analyses may increase the accuracy of diagnosis from cytological preparations. METHODS: Fifty-seven formalin-fixed paraffin-embedded (FFPE) samples were collected for next-generation sequencing (NGS) targeting potential driver mutations including TET2, DMN3TA, IDH2, RHOA, STAT3, and STAT5B. Competitive allele-specific TaqMan polymerase chain reaction (cast-PCR) was performed to validate the mutation status by using FFPE and preoperative fine needle aspiration cytology (FNAC) samples. RESULTS: Among these six candidate genes, only IDH2 and RHOA mutations were significantly more frequent in nodal subtypes, angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) (P = .002 and <0.001, respectively). All genes exhibited different mutation patterns except RHOA with a hotspot mutation involving the Gly17 residue. The RHOA G17V mutation was found in 15 (75%) of 20 AITL and two (22%) of nine PTCL, NOS. Cast-PCR using FFPE samples showed 100% concordance with NGS. Among 12 lymph node aspirates, the preliminary diagnoses were suspicious for lymphoma (3, 25%), atypical lymphoid cells (3, 25%) and benign/negative (6, 50%). Cast-PCR detected the RHOA G17V mutation in six (75%) of eight RHOA-mutated aspirates and revealed negative results in all (100%) of four wild-type aspirates, with an 83.3% (10/12) concordance comparing to FFPE samples. CONCLUSIONS: The RHOA G17V mutation serves as a useful biomarker for cytological assessment in AITL. The use of cast-PCR is valuable in the diagnosis of malignant lymphomas from cytological preparations, and thus avoiding the potential risks of invasive procedures.

Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma.

In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5-2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX.

Tbo-Filgrastim versus Filgrastim during Mobilization and Neutrophil Engraftment for Autologous Stem Cell Transplantation.

There are limited data available supporting the use of the recombinant granulocyte colony-stimulating factor (G-CSF), tbo-filgrastim, rather than traditionally used filgrastim to mobilize peripheral blood stem cells (PBSC) or to accelerate engraftment after autologous stem cell transplantation (ASCT). We sought to compare the efficacy and cost of tbo-filgrastim to filgrastim in these settings. Patients diagnosed with lymphoma or plasma cell disorders undergoing G-CSF mobilization, with or without plerixafor, were included in this retrospective analysis. The primary outcome was total collected CD34(+) cells/kg. Secondary mobilization endpoints included peripheral CD34(+) cells/µL on days 4 and 5 of mobilization, adjunctive use of plerixafor, CD34(+) cells/kg collected on day 5, number of collection days and volumes processed, number of collections reaching 5 million CD34(+) cells/kg, and percent reaching target collection goal in 1 day. Secondary engraftment endpoints included time to neutrophil and platelet engraftment, number of blood product transfusions required before engraftment, events of febrile neutropenia, and length of stay. A total of 185 patients were included in the final analysis. Patients receiving filgrastim (n = 86) collected a median of 5.56 × 10(6) CD34(+) cells/kg, compared with a median of 5.85 × 10(6) CD34(+) cells/kg in the tbo-filgrastim group (n = 99; P = .58). There were no statistically significant differences in all secondary endpoints with the exception of apheresis volumes processed (tbo-filgrastim, 17.0 liters versus filgrastim, 19.7 liters; P < .01) and mean platelet transfusions (tbo-filgrastim, 1.7 units versus filgrastim, 1.4 units; P = .04). In conclusion, tbo-filgrastim demonstrated similar CD34(+) yield compared with filgrastim in mobilization and post-transplantation settings, with no clinically meaningful differences in secondary efficacy and safety endpoints. Furthermore, tbo-filgrastim utilization was associated with cost savings of approximately $1406 per patient utilizing average wholesale price.

Subcutaneous panniculitis like T cell lymphoma associated with erythromelalgia.

Erythromelalgia is a rare disorder that simulates a small fiber neuropathy and patients often have painful erythematous extremities during episodes. It is of two types: A primary or inherited form that is sometimes associated with a Na channel mutation or a secondary disorder associated with an underlying systemic disorder. We present a 19-year-old boy who presented to us with erythromelalgia and a febrile illness with systemic rash. Detailed work-up revealed another unusual condition: Subcutaneous panniculitis like T cell lymphoma (SPTCL). This is the first report of an association of erythromelalgia with SPTCL.

Midtreatment ¹8F-FDG PET/CT Scan for Early Response Assessment of SMILE Therapy in Natural Killer/T-Cell Lymphoma: A Prospective Study from a Single Center.

UNLABELLED: In a prospective study of newly diagnosed or relapsed histologically proven extranodal natural killer/T-cell lymphoma (ENKTL) patients, we aimed to determine the accuracy of midtreatment (18)F-FDG PET for response assessment using both visual and quantitative analyses. METHODS: Twenty-four patients (12 men, 12 women; median age, 50 y; age range, 16-83 y) were referred for pre-, mid- (after 2-3 cycles of SMILE [prednisolone, methotrexate, ifosfamide, L-asparaginase, etoposide] chemotherapy), and end-treatment PET/CT scans (n = 24, 24, and 17, respectively) using a standardized protocol. Sixty-five PET/CT scans were analyzed visually using the Deauville 5-point score (DS), and the lesion with the highest maximum standardized uptake value (SUV(max)) was recorded. Survival curves were obtained using Kaplan-Meier analysis and compared using the log rank test, followed by multivariate analysis using the Cox proportional hazards model to assess the independent effects of International Prognostic Index (IPI) score (0-1 vs. 2-5), stage (stage I/II vs. stage III/IV), sex, DS (1-3 vs. 4-5), SUV(max), and change in SUV(max) on overall survival (OS) and progression-free survival (PFS). The mean (±SD) follow-up period was 32 mo (±21 mo). RESULTS: For 2-y OS, the following parameters were predictive: IPI score (P = 0.047), DS at mid- and end-treatment (P < 0.001), and SUV(max) at mid- and end-treatment (P < 0.001 and 0.045, respectively). For 2-y PFS, the following parameters were predictive: sex (P = 0.006), stage (P = 0.034), IPI score (P = 0.038), DS at mid- and end-treatment (P < 0.001 and 0.001, respectively), and SUV(max) at midtreatment (P = 0.001). Multivariate analysis showed DS on mid- and end-treatment scans to be the only significant independent predictor of both OS (P = 0.004 and 0.018, respectively) and PFS (P = 0.004 and 0.014, respectively). The 2-y estimate for OS and PFS was 81% and 62%, respectively, in patients with a DS of 1-3, compared with 17% in patients with a DS of 4-5 (P < 0.001 and 0.001, respectively). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the midtreatment DS for prediction of OS and PFS were 63%, 94%, 83%, 83%, and 83%, respectively. CONCLUSION: Midtreatment PET/CT is a valuable tool for early treatment response assessment in extranodal natural killer/T-cell lymphoma patients.

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.

The role of ¹8F-fluorodeoxyglucose positron emission tomography at response assessment after autologous stem cell transplantation in T-cell non-Hodgkin's lymphoma patients.

Although a few studies have evaluated the utility of ¹8F-fluorodeoxyglucose positron emission tomography (FDG-PET) in treatment-naive T-cell non-Hodgkin's lymphomas (T-NHLs), a few studies had been conducted to evaluate the role of FDG-PET in patients after treatment. A total of 41 patients with T-NHLs were included who underwent post-autologous stem cell transplantation (ASCT) PET for response assessment in addition to contrast-enhanced CT. The impact of post-ASCT PET on response assessment and predicting prognosis was retrospectively evaluated. Of the 41 patients, 11 (26.8%) patients showed discordant response between two image modalities (Cohen's κ = 0.465). The additional PET study actually guides changing the post-ASCT response in six (54.5%) of these 11 patients. Moreover, positive post-ASCT PET was associated with worse prognosis in event-free survival and overall survival than negative post-ASCT PET (P = 0.003 and P = 0.044, respectively). Excluding four patients in whom further confirmation was not available due to early mortality, in 22 lesions showing discrepancy between two image modalities, 12 lesions were true positive (N = 4) or true negative (N = 8) for PET and ten lesions were false positive (N = 7) or false negative (N = 3). The accuracy for the discrepant lesions was 54.5% (12 of 22), the overall accuracy for the detected lesions was 76.7% (33 of 43), and the overall accuracy for patients was 89.2% (33 of 37). Post-ASCT PET was useful in response assessment after ASCT, and the positive post-ASCT PET result was associated with worse prognosis than the negative post-ASCT PET in patients with T-NHLs.

Assessment of gemcitabine, cisplatin and methylprednisolone (GEM-P) combination treatment for non-Hodgkin T cell lymphoma.

T cell lymphoma is rare with few dedicated studies and no consensus regarding optimal treatment. We undertook a retrospective hospital review to assess the efficacy of gemcitabine, cisplatin and methylprednisolone (GEM-P) combination therapy. Twenty-nine patients were followed up for a median duration of 28 months. Twenty-three patients received standard GEM-P. Due to hearing impairment, 3 patients had cisplatin substituted with carboplatin and 1 with oxaliplatin. In 2 cases, rituximab was added to GEM-P in view of the presence of EBV + B cell clones. Overall response rate (RR) [complete response (CR) + partial response (PR)] was 73 % (95 % CI range 54-86 %). 11/29 (38 %) achieved CR and 10/29 (35 %) had PR. In first-line treatment, 4/10 patients achieved CR and 4/10 had PR relating to a RR of 80 %. CR was seen in 4/9 (45 %), 2/8 (25) and 1/2 (50 %) patients treated in the second, third and fifth-sixth line respectively. Thus, GEM-P was found to be effective as first-line or salvage therapy in T cell lymphoma.

Flow cytometric immunophenotypic assessment of T-cell clonality by vß repertoire analysis in fine-needle aspirates and cerebrospinal fluid.

Flow cytometric T-cell receptor V(ß) repertoire analysis (TCR-V(ß)-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-V(ß)-R in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-V(ß)-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-V(ß) protein expression. Subsequently, an abbreviated, patient-specific TCR-V(ß)-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-V(ß) antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-V(ß)-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma.

A novel clonality assay for the assessment of canine T cell proliferations.

Polymerase chain reaction (PCR) based clonality assays are an important tool to differentiate neoplastic from reactive lymphocyte populations. A recent description of the canine T cell receptor γ locus identified a large number of formerly unknown genes, and determined the locus topology consisting of 8 cassettes with up to 3 variable (V) genes, 2 joining (J) genes and one constant (C) gene each. Given that these data were not available when existing canine T cell clonality assays were developed, it is likely that they will fail to detect a subset of clonal lymphocyte populations. The objective of this study was to gauge the potential of canine T cell clonality assays to detect all rearranged T cell receptor γ genes and to develop an improved clonality assay. The primer sequences of existing clonality assays were aligned to the reference sequences of all rearranged genes and genes were scored as to the likelihood of being recognized by a primer. All four assays likely recognized subgroup Vγ2 and Vγ6 genes but 3 out of 4 assays were unlikely to detect subgroup Vγ3 and Vγ7 genes. All assays likely recognized Jγx-2 genes, but only two assays were likely to detect most Jγx-1 genes. Two assays had forward primers located as close as four nucleotides to the junctional region. A new multiplex PCR was designed with all primers combined in a single tube. An alternative primer set allowed identification of variable gene usage through gene specific forward primers. The coverage of all rearranged genes facilitated the detection of multiple clonal rearrangements per neoplastic sample. The new assay detected clonal DNA at a concentration of 5% within polyclonal background but detection thresholds were dependent on the gene usage of clonal rearrangements as well as the position of the clonal peak in respect to the polyclonal background. The new multiplex assay recognized 12/12 (100%) of confirmed neoplastic samples as compared to 2/12 (17%) by an existing assay. On a series of 60 diagnostic samples the concordance rate of both assays was 41/60 (68.3%). In 14/60 (23.3%) of the cases, the new multiplex assay yielded a clonal result while the existing assay gave a non-clonal result. In 5/60 (8.3%) of cases, the new assay yielded a non-clonal result while the existing primer set gave a clonal result. These findings suggest that the new multiplex assay has an improved sensitivity over traditional assays and is suited to reduce the rate of false-negative results.

Flow cytometric immunophenotypic assessment of T-cell clonality by Vß repertoire analysis: detection of T-cell clonality at diagnosis and monitoring of minimal residual disease following therapy.

Flow cytometric T-cell receptor (TCR)-V(ß) repertoire analysis (TCR-V(ß)-R) is a sensitive method for detection of T-cell clonality; however, no uniform approach exists to define clonality in neoplastic T cells. TCR-V(ß)-R was evaluated in patients with a diagnosis of T-cell neoplasia in initial diagnostic specimens from 41 patients and for minimal residual disease (MRD) monitoring in 61 sequential samples from 14 patients with mature T-cell neoplasia. Gating strategies and criteria for detection of T-cell clonality were determined. In all 41 initial specimens, T-cell clonality was demonstrated via TCR-V(ß)-R. The frequency of V(ß) usage was consistent with random neoplastic transformation of TCR-V(ß) subsets. MRD was successfully detected in follow-up samples from all 14 patients evaluated, Furthermore, MRD after therapy was quantitated in 48 peripheral blood specimens. TCR-V(ß)-R analysis is a sensitive method for detection of T-cell clonality and is useful for diagnosis and MRD detection in multiple specimen types.