TCR-γ Gene Rearrangement Research Articles

A-209 Utility of TCR-β and TCR-γ gene rearrangement to distinguish between tumour-stage mycosis fungoides and early-stage mycosis fungoides associated with CD30+ lymphoproliferative disorders

A-209 Utility of TCR-β and TCR-γ gene rearrangement to distinguish between tumour-stage mycosis fungoides and early-stage mycosis fungoides associated with CD30+ lymphoproliferative disorders

Isolated Monoclonal T-Cell Receptor Gene Rearrangement in a Lung Adenocarcinoma Harboring MET Exon 14 Skipping: Diagnostic Pitfall.

In the diagnostic workup of poorly differentiated tumors, T-cell receptor (TCR) clonality has long been considered as evidence of T-cell lymphoma. MET exon 14 skipping (METex14) is a mutation typically seen in lung adenocarcinoma. Herein, we present the first report of METex14 lung adenocarcinoma with isolated monoclonal TCRγ gene rearrangement. A 69-year-old woman presented to an outside hospital with pleural effusions. A pleural decortication demonstrated malignant cells positive for CD30 and CD138 but negative for BerEP4, KRT5, and EMA. An equivocal HHV8 staining was interpreted as positive, leading to the erroneous outside diagnosis of primary effusion lymphoma. Additional workup at our institution revealed a lack of HHV8 and T-cell markers but the presence of TCRγ clonality, pankeratin, and TTF1 expression. Repeat TCRγ testing on the in-house biopsy was negative for clonality. Next-generation sequencing detected METex14, confirming the diagnosis of lung adenocarcinoma. The potential diagnostic pitfall and prognostic/predictive implications are discussed.

Gamma-delta T-cell large granular lymphocytic leukemia in the setting of rheumatologic diseases.

T-cell leukemia originating from large granular lymphocytes (T-LGL leukemia) is a rare lymphoid neoplasia characterized by clonal proliferation of large granular T lymphocytes expressing αβ or γδ T-cell receptor (TCR) on the cell membrane. γδT-LGL leukemia, accounting for approximately 17% of all T-LGL leukemia cases, is associated with autoimmune diseases. However, the features of γδT-LGL leukemia in patients with rheumatologic diseases are still insufficiently characterized. In this retrospective study, 15 patients with rheumatologic disease-associated γδT-LGL leukemia were included. The patients were obtained from a single center from 2008 to 2023. Data related to clinical characteristics and rheumatologic diagnoses were collected. Immunophenotype evaluations as well as T-lymphocyte clonality (based on TCR-γ, TCR-β, and TCR-δ gene rearrangements), and signal transducer and activator of transcription (STAT) three and STAT5B mutation analyses (by next-generation sequencing) were performed on blood, bone marrow, and spleen samples. All but one patient had rheumatoid arthritis (RA). In 36% of patients, manifestations of γδT-LGL leukemia were present before or concurrently with clinical manifestations of RA. Splenomegaly was observed in 60% of patients and neutropenia (<1.5 × 109/L) was detected in 93% of cases. CD4-/CD8- and CD4-/CD8+ subtypes were detected in seven cases each. Mutations in STAT3 were detected in 80% of patients; however, STAT5B mutations were not detected. Evaluations of T-cell clonality and variant allele frequencies at STAT3 in the blood, bone marrow, and spleen tissue revealed an unusual variant of CD4-/CD8- γδT-LGL leukemia with predominant involvement of the spleen, involvement of the bone marrow to a less extent, and no tumor cells in peripheral blood. The mechanism by which γδT-LGL leukemia may induce the development of RA in some patients requires further investigation. Cases of RA-associated γδT-LGL leukemia with neutropenia and splenomegaly but no detectable tumor-associated lymphocytes in peripheral blood (the so-called splenic variant of T-LGL leukemia) are difficult to diagnose and may be misdiagnosed as Felty syndrome or hepatosplenic T-cell lymphoma.

T-cell receptor gamma gene rearrangement analysis of classic Hodgkin lymphoma using a BIOMED-2 assay: a paraffin-embedded tissue analysis of one hundred cases.

In the new WHO classifications of haematolymphoid tumours (WHO-HAEM5), classic Hodgkin lymphoma (cHL) is categorized into B-cell lymphoid proliferations and lymphomas. Although the majority of Hodgkin Reed-Sternberg (HRS) cells are of germinal center B-cell origin with some defects of B-cell transcription factors, they rarely express T-cell antigens or cytotoxic molecules. Clonality analyses on cHL samples using BIOMED-2 have been reported by several groups; however, those studies were only focused on Ig regions, including IgH, Ig-kappa, and Ig-lambda, and TCR-γ clonality analysis of cHL has not yet been explored. Here, we investigated TCR-γ gene rearrangement for one hundred cases using a PCR-based method. Four of one hundred (4%) cases showed TCR-γ clonal peaks. Of these, three were at an advanced stage and one patient died of the disease. To clarify whether HRS cells showed T-cell clonality or not, we performed PCR analysis using DNAs of microdissected HRS cells. Three samples showed identical clonal peaks with bulk specimens. Our results indicate that cHL is a heterogeneous disease of mainly B-cell and rarely T-cell origin with a special phenotype. Further molecular studies are warranted.

ALL-312 Ig/T-Cell Receptor Clonality Testing as a Method for Minimal Residual Disease Monitoring in T-Cell Acute Lymphoblastic Leukemia Patients

Assessment of early response to therapy by minimal residual disease (MRD) monitoring in T-cell acute lymphoblastic leukemia (T-ALL) has been proven to be a fundamental tool for guiding further therapy. Polymerase chain reaction (PCR)-based immunoglobulin (Ig)-T-cell receptor (TCR) clonality testing is a standardized technique for routine diagnosis of lymphoproliferation. It is also widely applied for MRD detection in T-ALL and is most suitable for patients without additional rearrangements detected. To present the clinical significance and utility of a molecular-based approach for post-induction MRD detection in patients with T-ALL. Single-center, descriptive, retrospective study including 11 adult patients with T-ALL treated at the University Clinic for Hematology - Skopje from January 2018 to January 2022. TCR gene analyses were performed in 11 adults with T-ALL. TCR rearrangements in bone marrow samples were analyzed by multiplex fluorescent PCR amplifications of the TCRB-Vβ-Jβ, Dβ-Jβ, TCRG-Vγ-Jy, TCRD-Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ, and Dδ-Jδ regions and subsequent capillary electrophoresis of PCR products by automatic DNA analyzer, following EuroClonality/BIOMED-2 guidelines. MRD status was assessed at the end of the induction cycle. Patients received a BFM-based protocol or Hyper-CVAD/high-dose methotrexate (HDM) and cytarabine (ARA-C) regimen. TCR rearrangements were detected in all 11 patients. All patients were negative for additional molecular prognostic markers. TCRB and TCRG gene rearrangements were slightly dominant over TCRD (64.3% vs. 50%). A total of 6 adolescents and young adults (mean age: 24 years) were treated with BFM. At the end of the induction regimen, MRD negativity was obtained in 5 patients (83.3%) and was sustained up to 3 months from stem cell transplantation (SCT) in 3 patients. The other 2 patients died from CNS infiltration. Hyper-CVAD/HDM-ARA-C was administered in 5 patients (mean age: 49 years). One patient died during the induction protocol due to intracerebral hemorrhage. Among the others, CR at the end of the first cycle was achieved in 3 patients, and a total of 4 cycles were applied. Two patients proceeded to SCT. The molecular technique for testing Ig/TCR clonality is a useful tool for post-induction MRD monitoring in patients with ALL, directing the further approach and clinical decisions.

Characteristics and prognosis of children with recurrent T-cell acute lymphoblastic leukemia: a long-term follow-up report in China

This study assessed the relapse characteristics and prognosis of 145 children newly diagnosed with T-cell acute lymphoblastic leukemia (T-ALL). The overall complete response (CR) rate was 91.7% (133/145), and the overall recurrence rate was 31.6% (42/133). The recurrence rate in the intermediate-risk (IR) group and high-risk (HR) group was 15.4% and 47.1%, respectively (p < 0.001). Patients with young age, early T-cell precursor ALL, central nervous system (CNS) involvement, TCRγ gene rearrangement, karyotypic abnormalities, or absence of TCRβ gene rearrangement (p < 0.05) tended to relapse. All recurrences occurred within 36 months after diagnosis. The HR group recurred earlier than the IR group (p= 0.026). The 3-year overall survival (OS) rate was significantly lower in the HR group than in the IR group (p < 0.001). All relapsed children died within 12 months after recurrence. Early intervention may benefit children with a high risk of recurrence.

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.

Self-regressing oral CD30-positive, EBV-negative, T-cell lymphoproliferative lesions. A poorly understood process highlighted by ominous clinicopathologic features and indolent behavior

Intraoral, primary, CD30-positive (CD30+) T-cell lymphoproliferative disorders (TLPDs) are uncommon, and their clinicopathologic presentation and management can vary and may be challenging. Herein, we present a retrospective study of 4 examples of self-regressing primary CD30+ TLPD affecting the gingiva. Archived files were retrospectively reviewed for oral CD30+ TLPDs featuring (1) proper immunohistochemical documentation, (2) Epstein-Barr virus negativity, (3) adequate follow-up information corroborating regression, and (4) no history of hematopoietic malignancy or related-mucocutaneous disease. Three women and 1 man (age range, 55-82 years; mean, 68.3 years) presented with rapidly growing gingival ulcers. Microscopic evaluation revealed diffuse infiltration by sheets of large, atypical cells admixed with lymphocytes and eosinophils, showing angiocentric distribution, focal neurotropism, and muscle infiltration. The lesional cells consistently stained for CD3 and CD30 and were variably immunoreactive against CD2, CD4, CD5, CD7, and CD8, but were negative for ALK1 and EBV-encoded small RNA. TCR-γ gene rearrangement studies revealed a monoclonal T-cell population in 1 case. All lesions showed complete regression 2 to 8 weeks postoperatively (mean follow-up, 4.5 weeks). Notwithstanding their alarming clinicopathologic appearance, there are CD30+ TLPDs confined to the oral cavity that have an indolent course. However, clinical staging is essential to exclude aggressive systemic malignancy.

STAT3 Mutations and Anemia with Pure Red Cell Aplasia Are the Distinctive Features of T Cell Receptor Gamma Delta Type Large Granular Lymphocyte Leukemia

Large granular lymphocytic leukemia (LGLL) is a heterogenous group of disorders. The clinical presentations of LGLL are highly variable; it may present with neutropenia or anemia, complicated with recurrent infections, autoimmune diseases such as rheumatoid arthritis, and neurological symptoms, requiring therapeutic interventions. It may present only with indolent laboratory changes without any symptoms. The cytological phenotypes of LGLL also vary and they commonly include CD8+TCR αβ-type, CD4+TCR αβ-type, and NK cell-type. NK cell-type LGLL is designated as chronic lymphoproliferative disorder of NK cells (CLPD-NK). TCR γδ-type LGLL (γδT-LGLL) is even rarer and it often confers cytopenia. The genetic backgrounds of LGLL with CD8+TCRαβ-type, CD4+TCRαβ-type and NK cell-type have been elucidated with the recent sequencing technologies. JAK/STAT signaling genes and chromatin-modifying genes were frequently mutated in the non-leukemic counterpart of the lymphoid malignancies with γδ TCR immunophenotypes, such as hepatosplenic T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma and primary cutaneous gamma delta T-cell lymphoma. However, the genetic basis of γδT-LGLL and the genetic-phenotypic associations remain unclear. The aim of this study is to identify clinical and genetic characteristics of γδT-LGLL in comparison with other LGL leukemias. In total, 56 LGLL patients were recruited; six patients with γδT-LGLL, 25 CD8+ TCRαβ type, 8 CD4+TCR αβ type and 17 CLPD-NK, in this study. All cases were negative for EBV. The median age of patients with γδT-LGLL was 48 years old and five of them were male. The median LGL counts and hemoglobin level were 1.81 ×109/L (range; 0.60-4.42) and 5.8 g/dL (range; 3.0-11.2), respectively. All patients with γδT-LGLL had anemia and/or neutropenia, although no patients were thrombocytopenic. 4 patients presented with pure red cell aplasia. Common immunophenotypes of γδT cells were CD2+ CD3+ CD4-CD7+. 3 patients were CD8+. CD16, CD56 and CD57 were positive in 3, 0, and 1 patient, respectively. γδT cells of all patients showed a monoclonal pattern of TCRγ gene rearrangements with BioMed 2 protocol. We performed amplicon sequencing of entire coding regions of STAT3, STAT5B and TNFAIP3 genes and allele-specific PCR for several hot spot regions of STAT3 and STAT5B genes using extracted DNA from mononuclear cells of peripheral blood. Mutations in STAT3 and TNFAIP3 were identified in 100% (6/6) and 17% (1/6), respectively. Among the10 STAT3 mutations identified, 5 were located in the SH-2 domain, while other 3 were in coiled-coiled domain and the other 2 were in the DNA binding domain. The median variant allele frequencies of STAT3 was 2.0% (0.7-22.6) and STAT3 mutations were restricted to sorted γδT cells in all 3 patients examined. No patient was positive for STAT5B mutations, including N642H and Y665F with allele specific PCRs. One of 5 patients who were treated with cyclosporin A was refractory. Two patients were treated with cyclophosphamide, showing clinical improvements. The patients with γδT-LGLL were younger than those with other types of LGLL (P=0.048) and anemia and STAT3 mutations were significantly more frequent in γδT-LGLL (P value; 0.003 and 0.02, respectively). PRCA was a consistent cause of anemia in γδT-LGLL. Low frequency of STAT5B mutations among γδT-LGLL patients was in contrast to other aggressive γδT cell lymphoma in which STAT5B frequently mutated. The clinical characteristics of γδT-LGLL patients may be defined by younger age and the anemia due to pure red cell aplasia, and the frequent STAT3 mutations might be a novel molecular marker for γδT-LGLL. Disclosures Yamane: Pfizer: Research Funding; Celgene: Honoraria; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Astellas Pharma: Research Funding; Eli Lily and Company: Research Funding. Ishida:Eli Lily and Company: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Celgene: Honoraria.

Indeterminate Cell Histiocytosis and Mycosis Fungoides. A Hitherto Unreported Association

Indeterminate Cell Histiocytosis and Mycosis Fungoides. A Hitherto Unreported Association

QUANTITATIVE RHOA Gly17Val ALLELE-SPECIFIC POLYMERASE CHAIN REACTION AND T-CELL CLONALITY ANALYSIS IN ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA

Introduction. Аngioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma, characterized by abundant polymorphocellular infiltrate of lymph nodes with the small number of tumor CD4 + T-cells. AITL could often be misdiagnosed as reactive processes and other lymphomas, including Hodgkin’s lymphoma. Molecular methods of determining clonality are used to confirm the diagnosis. TCR gene rearrangements detected in the lymph nodes may not coincide with those detected in the bone marrow (BM), blood or skin, that makes their interpretation difficult. Recently discovered point somatic RHOA (Ras homolog gene family, member A) Gly17Val mutation is present in 53–71 % of angioimmunoblastic T-cell lymphomas. The aim of the study was to evaluate the number of RHOA Gly17Val mutated cells in different tissues of AITL patients and to correlate it with corresponding T-cell clonality results. Materials and methods . TCRG and TCRB gene rearrangements were PCR-amplified according to BIOMED-2 standardized protocol and analyzed by capillary electrophoresis on ABI Prism 3130. Gly17Val mutation was analyzed by quantitative allele-specific (qAS) TaqMan Real-Time PCR assay. Lymph nodes (LN) and skin biopsies, blood and BM samples were studied for 40 patients with AITL. Results. The clonal TCR gene rearrangements were found in 37 (92 %) of 40 patients in LN and in 26 (96 %) of 28 patients in BM, but they were not matched in length in LN and BM in 12 (46 %) of 26 patients. RHOA (Gly17Val) mutation in LN was revealed in 60 % (24 of 40) patients. Number of cells with RHOA mutation was highest in the LN (in average 26.7 % of the total cells), while in the BM RHOA mutation were undetectable (in 7 patients), or detected in 10 patients in a low quantity (in average 2 % of the total cells). Skin, blood and BM samples with the T-cell clonality peaks that differ from those found in the LN were also negative for the presence of cells having RHOA Gly17Val mutation. Also, in four patients we showed that clonal products in BM and blood that do not coincide with LN refer to CD8+ lymphocytes. Conclusions. The results of the study demonstrate that the quantitative determination of cells with the mutation of RHOA Gly17Val must be taken into account in staging the disease and interpreting the results of T-cell clonality assay. Clonal cells with rearrangements not matching those identified for the LN should be considered reactive.

Clonal CD57+ Cells in T-Cell Large Granular Lymphocytic Leukemia

Large granular lymphocytic leukemia (T-LGL) characterized by steady increase in large granular lymphocyte counts over 2*109/l in peripheral blood. T-LGL frequently manifested with splenomegaly, neutropenia, anemia, lymphocytosis, and complicated by infectious diseases. T-LGL cells have mature, post-thymic immune phenotype expressing CD3+,TCRab, CD8+, CD57+ etc.In up to 1/3 of cases T-LGL is combined with autoimmune diseases like rheumatoid arthritis (RA) and could be misdiagnosed asFelty'ssyndrome. Differential diagnosis requiresclonalitytesting. However admixture of polyclonal large granular lymphocytes may compromise PCR sensitivity and decrease the diagnostic value of the test. CD57 + is a typical, but not strictly specific marker of T-LGL cells. Therefore, enrichment in CD57+ cells may circumvent PCR sensitivity issues.This study aimed to assess the possibility and feasibility of clonal assay performed on a selected CD57+ population of peripheral blood lymphocytes in patients with T-LGL for differential diagnosis/verification.The study included 13 patients with RA and assumed T-LGL diagnosis. CD57+ lymphocytes were isolated by means ofimmunomagneticselection onMiltenyiBiotecbeads.T-cellclonalitywas assessed by T-cell receptor gamma chain TCRG (VG - JG) gene rearrangements using multiplex PCR with BIOMED-2 primer system and subsequent Gene-Scan analysis.Polyclonal pattern in bulk lymphocyte population was observed in patients ## 1-3 (Table 1). CD57+ enrichment allowed to determineclonalityand approve T-LGL diagnosis for patient #1 (Figure1). Due to the lack ofclonalityin CD57+ population T-LGL diagnosis was excluded for patient #2. Patient #3 showedoligoclonalpattern in CD57+ enriched population.Bulk lymphocyte population in 5 of 13 patients was monoclonal. After CD57+ enrichment 3 of these 5 patients showed identical monoclonal pattern. Other two patients aquiredadditional peaks (Figure2). In 3 of 13 patients monoclonal peak exceeded the polyclonal background less than 3 times and was interpreted as doubtful monoclonal result. After CD57+ enrichment oligoclonalpattern was detected in these cases. 2 of 13 patients preserved oligoclonalpattern for both bulk and CD57+ enriched lymphocyte populations.CD57+ selection was supportive for diagnostic procedure in 2 cases so far. Based on CD57+ population analysis in one case T-LGL diagnosis was approved while in other excluded. All other included cases gained no benefit from CD57+ enrichment. Frequentoligoclonalpattern in T-LGL may reflect restriction of TCR repertoire of effector cells.We conclude that CD57+ enrichment forclonalitystudies in T-LGL is of limited value for diagnostic procedure in clinical practice. At least at the present level of understanding of T-LGL pathogenesis and reactive lymphocyte populations involved. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple Clonal TCR Gene Rearrangements Are Typical in Peripheral T-Cell Lymphoma Not Otherwise Specified

Introduction: Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a heterogeneous subgroup of PTCL with no consistent immunophenotypic, genetic and clinical features. PTCL-NOS represents less than 29% of all T-cell lymphomas in adults and often has an aggressive course with extranodal and systemic involvement. This study is focused on the application of clonality testing methods for PTCL-NOS diagnosis and monitoring.Patients and Methods: Biopsies, blood and bone marrow samples were tested for 30 patients with PTCL-NOS: the ratio of male/female was 15/15, median age was 56 years (32-75), and 22 of 30 patients had IV stage of the disease. TCRG and TCRB gene rearrangements were evaluated for T cell clonality analysis. We used PCR-based methods (BIOMED-2 standardized clonality protocol) followed by GeneScan analysis on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).Results: Clonal TCR gene rearrangements were found in 29 of 30 patients (97%): TCRG in 27 of 30 (90%) and TCRB in 29 of 30 (97%). Primary biopsies or bone marrow samples showed oligoclonal pattern of rearrangements (more than 3 clonal peaks in TCRG or TCRB GeneScan profiles) in 13 of 30 patients (43%). New clonal rearrangements in affected tissues (skin, tonsil, CSF, lymph nodes and etc.) during disease progression were found in 15 of 24 (63%) patients. Various representations of clonal rearrangements in different tissues indicate the presence of several tumor clones in the majority of cases (19 of 30 patients; 63%). The presence of several clones correlated with the number of tissues examined (rs = 0.6, p <0.0005). The detection rate of additional clonal rearrangements was 90% (9 of 10 patients) when 3 and more tissues were tested. The number of clonal rearrangements was not correlated with age (p = 0.43) or the stage of disease (p = 0.29). One can speculate that the presence of multiple clones in the PTCL-NOS could reflect genetic instability of this tumor.Conclusion: Oligoclonality, clonal evolution and the appearance of new clones in the progression of the disease are typical features of PTCL-NOS. Multiple clonal rearrangements complicate diagnostics and assessment of minimal residual disease in this disease. DisclosuresNo relevant conflicts of interest to declare.

Clonal Rearrangements and Malignant Clones in Peripheral T-cell Lymphoma

To assess the feasibility and informative value of T-cell clonality testing in peripheral T-cell lymphoma (PTCL). Biopsies of involved sites, blood, and bone marrow samples from 30 PTCL patients are included in the study. Rearranged TCRG and TCRB gene fragments were PCR-amplified according to the BIOMED-2 protocol and analyzed by capillary electrophoresis on ABI PRISM 3130 (Applied Biosystems). TCRG and TCRB gene clonality assay was valuable in confirming diagnosis in 97% of PTCL patients. T-cell clonality assay performed on blood or bone marrow samples reaffirmed lymphoma in 93% of cases, whereas morphological methods were informative in 73% of cases only. We observed multiple TCRG and TCRB gene rearrangements, loss of certain clones in the course of the disease, as well as acquisition of new clones in 63% of PTCL cases, which can be attributed to the genetic instability of the tumor. TCRG and TCRB gene clonality assay is beneficial for the diagnosis of PTCL. However, the presence of multiple clonal rearrangements should be considered. Clonal evolution in PTCL, particularly acquisition of new clones, should not be treated as a second tumor. Multiple TCRG and TCRB gene rearrangements may interfere with minimal residual disease monitoring in PTCL.

Monitoring minimal residual disease by qualitative detection of immunoglobulin H/T cell receptor γ by using multiplex polymerase chain reaction in children with acute lymphocytic leukemia

Objective To explore the prognostic significance in monitoring minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) by a simple method, and to detect cloned immunoglobulin H (IgH) and T cell receptor γ (TCRγ) gene rearrangements by using multiplex polymerase chain reaction (PCR) and automated fragment analysis. Methods Bone marrow samples were collected from 86 newly diagnosed cases of childhood ALL at the Department of Pediatrics, the First Affiliated Hospital of Sun Yat-Sen University, from May of 2009 to August of 2013.IgH and TCRγ gene rearrangements were amplified by qualitative multiplex PCR.The clonality of PCR production was analyzed by GENEMAPPERID software.Only those carried monoclonal IgH/TCRγ on diagnosis were arranged to monitor MRD.Detectable monoclonal IgH/TCRγ by the end of induction was defined as MRD positive.All patients were treated with GD2008 ALL protocol.Clinical data of all newly-diagnosed ALL patients in the corresponding period were reviewed.The final follow-up on May 31, 2014.Survival rates and event free survival (EFS) curves were estimated by the Kaplan-Meier, and compared by using the log-rank test. Results The percent age of 94.2(81/86 cases) patients was at least 1 marker positive.Subsequent MRD was monitored in 79 cases.The median follow-up time was 20 months (9-61 months). By the end of induction, 20 cases were MRD positive and 59 cases were MRD negative, and the 3-year EFS were 56.4%±14.7% and 94.0%±3.4% (χ2=8.563, P=0.003), respectively.According to the traditional prognostic stratification criteria, MRD was detected 65 cases in the non-high risk group: 23 cases in stan-dard risk group and 42 cases in intermediate risk group, and the difference of 3-year EFS had no statistical significance (95.3%±4.7% vs 76.6%±9.0%, χ2=0.934, P=0.334). While using MRD by the end of induction as a risk stratification criterion, there was a statistical significant difference compared with the 3-year EFS for MRD-negative (n=52) group and MRD-positive (n=13) group(93.1%±3.8% and 59.5%±16.2%, χ2=7.128, P=0.008). Conclusions It is a simple but feasible method to monitor MRD in childhood ALL by using this qualitative multiplex PCR with automated fragment analysis for monoclonal IgH/TCRγ gene rearrangements.MRD by the end of induction can be used as a more accurate risk stratification criterion than the traditional one.It is worth of further research. Key words: Acute lymphoblastic leukemia; Prognosis; Minimal residual disease

Clinicopathological study of pulmonary extranodal nature killer/T-cell lymphoma, nasal type and literature review

ObjectivesMalignant tumors of the lung are predominantly derived from epithelial tissue, such as squamous cell carcinoma and adenocarcinoma, while pulmonary lymphatic and hematopoietic tumor is relatively rare. Extranodal NK/T-cell lymphoma (ENKTL), nasal type, originates in nasal and extra-nasal sites anatomically. This distinct non-Hodgkin lymphoma is endemic and is characterized by a highly aggressive clinical course and dismal survival outcome. Extra-nasal ENKTL, especially pulmonary ENKTL, is rare compared with nasal type ENKTL and has received relatively little attention. Therefore, this study was conducted to assess the clinicopathological features of pulmonary ENKTL and to promote awareness of this malignancy. Materials and methodsAll cases of ENKTL, nasal type diagnosed from January 2008 to June 2014 in our institution were collected, and those with pulmonary involvement were selected for further study. The eligible cases were analyzed retrospectively: medical recordings, imaging manifestations, pathological features, immunophenotypes, EBER1/2 hybridizations in situ and other related literatures were reviewed. Results and conclusionsA total of 1105 cases were diagnosed as ENKTL, nasal type, in this period, and 8 cases (7.2‰) had lung involvement. Seven cases had core biopsy, and for 1 case, a resected tissue specimen was available. The group was composed of 6 men and 2 women (gender ratio 3:1) with ages ranging from 19 to 44 (average age of 33.5) years. In this group, 2 cases were secondary and 5 cases were primary. The clinical symptoms and computed tomography (CT) manifestations were nonspecific. Histologically, the neoplasms presented angiocentric and angiodestructive growth patterns with different degrees of inflammatory response and necrosis. The neoplastic cell sizes were heterogeneous with spectra of small to large or mixed-composition types. For the immunophenotypes, all cases were positive for CD3ɛ and cytotoxic granule (granzyme B or TIA-1). The positive ratios of CD56 and CD30 were 6/8 and 4/5 respectively. All 8 cases showed positive in situ hybridization for Epstein–Barr virus-encoded small RNA (EBER). TCR-γ gene rearrangement was tested in 4 cases, and only 1 of these cases was monoclonal. Laboratory testing demonstrated that the whole blood was decreased while the average level of LDH was elevated. Six bone marrow biopsy specimens were negative and showed no neoplastic cells infiltration. For treatment, 4 individuals accepted chemotherapy and 1 patient underwent localized tumor resection surgery. The follow-up information was available for 6 patients, 1 of whom was alive and the other 5 cases survived shortly between 20 days and 4 months. ENKTL, nasal type of lung is very rare, and the diagnosis is challenging due to nonspecific clinical symptoms and imaging results. The diagnosis of pulmonary ENKTL should be based on comprehensive clinical, imaging, histopathological and molecular examination. More effective treatment strategies are required for this disease.

Construction of a multicolor GeneScan analytical system to detect clonal rearrangements of immunoglobulin and T cell receptor genes in canine lymphoid tumors

Polymerase chain reaction (PCR) amplification to detect immunoglobulin heavy chain (IgH) and T cell receptor γ-chain (TCRγ) gene rearrangements has recently become widely used as part of the diagnostic strategy for lymphoid tumors in dogs. In this study, we constructed a multicolor GeneScan analytical system to improve the sensitivity and resolution of the clonality analysis of antigen receptor gene rearrangements in dogs. We used 7 reactions per sample, with 2 PCR conditions, to amplify IgH/TCRγ and control genes. By using multicolor-labeled primers, these 7 PCR products could be combined into 3 tubes before capillary electrophoresis. Clonal rearrangement of the IgH/TCRγ genes was detected in 93.3% of dogs with multicentric lymphoma and 84.6% of dogs with gastrointestinal lymphoma. Detection sensitivity of the clonally expanded cells in the background of normal peripheral blood mononuclear cells was 1–10%. The multicolor GeneScan analytical system developed here may prove to be helpful for the diagnosis of lymphoid tumors in dogs.

Hepatosplenic γδ T-cell lymphoma after allogeneic bone marrow transplantation

Dear Editor, Hepatosplenic γδ T-cell lymphoma (HSγδTCL) is a rare extranodal T-cell non-Hodgkin lymphoma (NHL) that arises from a small subset of γδ T-cell receptor-expressing lymphocytes. It is characterized by an aggressive clinical course and poor response to conventional chemotherapy. HSγδTCL occurs in both immunocompetent and immunocompromised patients. To the best of our knowledge, it has never been observed after allogeneic bone marrow transplantation (alloSCT). Here, we report the case of a 47-year-old man who developed a HSγδTCL 10 years after an allo-SCT. The patient presented with fever, fatigue, and abdominal discomfort, 10 years after a familial allo-SCT performed for primary myelofibrosis. Physical examination showed marked hepatosplenomegaly in the absence of lymphadenopathy. Laboratory findings included mild thrombocytopenia (127, 000/mm) and leucopenia (2420/mm), but normal LDH and liver enzymes. Virological tests were negative for EBV, CMV, HIV, HBV, and HCV. The first bone marrow aspirate was normal. Liver biopsy disclosed sinusoidal infiltration by medium-sized lymphocytes with irregular nucleus that expressed a γδ T-cell phenotype (CD2+, CD3+, CD5−, CD4−, CD8−, CD7+, CD56+/−, γδTCR) and some cytotoxic granule-associated proteins (TIA1+, granzyme B−) (Fig. 1a–d). Molecular analysis showed a monoclonal TCRγ gene rearrangement and in situ hybridization (EBER) did not reveal the presence of EBV. FISH analysis demonstrated an isochromosome 7. The malignant T-cells were of host origin. The second marrow aspirate showed infiltration by phenotypically identical T-cells. The patient received three courses of cladribine with poor response, followed by myeloablative chemotherapy with busulfan-cyclophosphamide and a second allo-SCT from another sibling donor. He remained in complete remission for 61 months and relapsed. He failed to respond to belinostat, a histone deacetylase inhibitor, and died of refractory disease. Post-transplant lymphoproliferative disorders (PTLDs) are a common complication of immunosuppression after transplantation. The majority is of B-cell origin, with a strong association with EBV infection. The incidence is directly related to the intensity of immunosuppression. Reduction of immunosuppressive therapy may induce complete remission of the disease. In contrast, T-cell lineage PTLDs are very rare; they exhibit either a T-helper (CD4+) or a T-cytotoxic (CD8+) phenotype, and most of them express the αβ TCR. EBV is only detected in a minority of cases [1]. HSγδTCL accounts for less than 1 % of all NHL [2] and usually occurs in immunocompetent young adult males. Up to 20 % arise in the context of chronic immunosuppression, most commonly after solid organ transplantation, or neoplasms such as Hodgkin lymphoma and acute myeloblastic leukemia, but autoimmune disorders such as inflammatory bowel diseases, idiopathic thrombocytopenic purpura, and prolonged antigenic M.<C. Vekemans (*) : L. Michaux : E. Van Den Neste :A. Ferrant Department of Hematology, Cliniques universitaires Saint-Luc, Universite catholique de Louvain, 10 Avenue Hippocrate, 1200 Brussels, Belgium e-mail: marie-christiane.vekemans@uclouvain.be

Significance of detecting IgH and TCRγ gene rearrangements in patients with hemopoietic maligancies by real-time quantitative PCR.

The aim of our study was to investigate the association of IgH and TCRγ gene rearrangements in hematological malignancies with the disease and clinical application. IgH and TCRγ gene rearrangements were determined in 69 paraffin and bone marrow specimens with SYBR Green I fluorescent dye and RQ-PCR method, including 21 paraffin-embedded tissues of the onset cases and 48 bone marrow samples, representing 15 ALL and 25 AML cases. After chemotherapy, 8 cases were NHL; the 10 cases of the negative control group were healthy people. Among the ALL cases, the IgH rearrangement occurred in 80.0%, the TCRγ rearrangement in 46.7%, and both gene rearrangements in 46.7%. Among the AML cases, the IgH rearrangement occurred in 72.0%, the TCRγ rearrangement in 68.0%, and both gene rearrangements in 60.0%. In the lymphoma cases, the IgH rearrangement occurred in 93.1%, the TCRγ rearrangement in 51.7%, and both gene rearrangements in 44.8%. In the negative control group, the 10 cases were all negative. There was the phenomenon of "sequence-non-fidelity" in the hematologic malignancies; the detection rate of both genes was much higher than that of the single gene. The application of the RQ-PCR method in the detection of IgH and TCRγ gene rearrangements in hematologic malignancies has important clinical significance in MRD monitoring.

Clinicopathologic features and prognosis of T lymphoblastic lymphoma associated with Langerhans cell histiocytosis

To study the clinicopathologic features, immunophenotype and molecular genetic changes of T lymphoblastic lymphoma (T-LBL) associated with Langerhans cell histiocytosis (LCH). Three cases of T-LBL associated with LCH were included. The morphologic characteristics were reviewed along with immunohistochemical profiling using EnVision method and TCR gene rearrangement by PCR. A review of composite lymphoma previously reported in the literature was performed. All three patients were male with the mean age of 61.7 years. One was Hans and the other 2 were Uyguers. All presented with superficial lymph node enlargement. Biopsy of lymph node showed two abnormal cell populations: distended sinus by large, pale histiocytes with nuclear grooves, and the interfollicular region containing immature-appearing cells with irregular nuclei slightly larger than that of small lymphocyte, dispersed chromatin, inconspicuous nucleoli, scant cytoplasm, and scattered mitotic figures. These cells presented in aggregates and small sheets interspersed with normal-appearing lymphocyte. The histiocytes were positive for CD1a, S-100 protein and CD68. The lymphoma cells were positive for CD3, CD7, TdT and CD34. TCR-γ gene rearrangement was detected in one case by PCR technology. One case involved bone marrow with double phenotype acute leukemia. Amongst the 8 including 5 reported cases, there were 4 males and 4 females. The mean age of the patients and the median age were 54 years. Lymphoadenopathy was the most common presentation. Bone marrow was involved in 4 cases. The time of follow-up was 2 to 27 months. The median survival was 5.5 months and the one-year survival rate was 33.3%. Diagnosis of T-LBL and LCH should be based on typical morphology, immunophenotype and molecular genetic findings, with differential diagnoses including Langerhans cell hyperplasia originated from dermatopathic lymphadenopathy. When involving lymph node, extensive sampling supplemented by immunohistochemical staining is important to reach a correct diagnosis. Although coexistent T-LBL and LCH is clonally related, the understanding of its pathogenesis requires further investigation.