T-cell Receptor Gamma Rearrangements Research Articles

Molecular and clinicopathological features of granzyme B-negative extranodal NK/T-cell lymphoma

Extranodal NK/T-cell lymphoma (ENKTL) generally expresses cytotoxic molecules, including granzyme B (GZMB), T-cell-restricted intracellular antigen-1 (TIA-1), and perforin; however, the expression of these molecules varies across cases. We performed gene expression profiling and identified unique biological and clinicopathological features of GZMB-negative ENKTL. We reviewed the clinicopathological characteristics of 71 ENKTL samples. Gene expression profiling on nine ENKTLs using multiplexed, direct, and digital mRNA quantification divided ENKTLs into Groups A (n = 7) and B (n = 2) through hierarchical clustering and t-distributed stochastic neighbor embedding. Group B was characterized by downregulation of genes associated with IL6-JAK-STAT3 signaling and inflammatory responses. GZMB mRNA expression was significantly downregulated in Group B. GZMB protein expression was evaluated with immunohistochemistry in all 71 ENKTLs, and expression data of Tyr705-phosphorylated STAT3 (pSTAT3) and MYC from our previous study was utilized. T-cell receptor gamma (TRG) gene rearrangement in the selected samples was also assessed using PCR. GZMB expression was higher in pSTAT3-positive (p = 0.028) and MYC-positive (p = 0.014) ENKTLs. Eighteen percent (13/71) of all ENKTLs were negative for GZMB (defined by positivity <10 %); patients with GZMB-negative ENKTLs were often in a higher clinical stage (p = 0.016). We observed no other correlations with clinical parameters or TRG rearrangement and no significant association between GZMB expression and survival. In conclusion, GZMB expression is highly heterogeneous in ENKTLs and is associated with the activation of the JAK-STAT3 pathway and higher MYC expression. GZMB-negative ENKTLs correlate with an advanced clinical stage, suggesting the potential utility of GZMB immunohistochemistry as a biomarker of ENKTL.

Characterization of CD3+/CD20+ canine large-cell lymphoma.

Immunophenotyping of canine large-cell lymphoma (LCL) for B-cell and T-cell surface antigens is commonly performed to better predict the clinical outcome. Expression of surface antigen CD3 is associated with T-cell malignancies; surface antigen CD20 is expressed on B cells. However, a small subset of canine LCLs expresses both CD3 and CD20 (CD3+/CD20+); this form of lymphoma remains poorly defined at the molecular level. In a retrospective study, we aimed to better characterize immunophenotypic properties and antigen receptor clonality of CD3+/CD20+ LCL. We selected formalin-fixed, paraffin-embedded tissues from 10 cases of CD3+/CD20+ LCL and breed-matched controls of peripheral large T-cell lymphoma (PTCL) and diffuse large B-cell lymphoma (DLBCL). Using PCR for antigen receptor rearrangement (PARR), we identified monoclonal T-cell receptor gamma (TCRγ) rearrangements in all CD3+/CD20+ cases. Three of 10 cases had monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of cross-lineage rearrangement. There was no significant difference in the frequency of antigen receptor rearrangement between CD3+/CD20+ and PTCL cases. In comparison with DLBCL, CD3+/CD20+ LCL had TCRγ rearrangement more frequently and IgH rearrangement less frequently, respectively. Immunolabeling of the B-cell marker PAX5 occurred less frequently in all CD3+/CD20+ LCL cases compared to the DLBCL controls. Immunolabeling for BCL-2 was robust, regardless of immunophenotype. Nuclear Ki67 positivity was variable in CD3+/CD20+ cases, indicating a heterogeneity in proliferation. Overall, cases of canine CD3+/CD20+ LCL had properties similar to PTCL, suggesting a similar histogenesis of these 2 subsets.

Clonal T-cell proliferations occasionally occur in Kikuchi–Fujimoto disease

Kikuchi-Fujimoto disease (KFD) is a benign self-limiting disorder that frequently leads to swelling of cervical lymph nodes in young women. It has a characteristic histologic appearance with sharply demarcated foci containing apoptotic debris, histiocytes, and proliferating large T-cells. Since in the past years, core needle biopsies have been increasingly used for diagnostic work-up, a small biopsy of the pathognomonic proliferating T-cell foci may lead to misinterpretation as a large T-cell neoplasia. The aim of the present study therefore was to analyze how frequently clonal T-cell receptor (TCR) amplificates may be obtained in KFD using a commonly used TCR gamma rearrangement clonality assay. In 88 KFD cases, TCR gamma clonality assays could be successfully applied. Clonal peaks of TCR gamma in front of a polyclonal background were observed in 15 cases (18%). The investigated clinical parameters (age, gender, extent of infiltration of the lymph node, percentage of proliferative compartment) did not differ between patients with detectable TCR gamma clones from those patients who had polyclonal TCR gamma results. Our study therefore demonstrates that clonal TCR gamma amplificates may be obtained in any type of KFD and that an over-interpretation of clonal T-cell proliferates in diagnostically equivocal material should be avoided.

Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia.

Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

Abstract 5233: MEF2C dysregulation is associated with immature T cell immunophenotype, absence of biallelic deletion of TCR-gamma and inferior survival

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy affecting both children &amp; adults. Unlike B-ALL, the prognostic markers are not clearly defined in T-ALL. In this study, we evaluated the correlation of MEF2C gene expression in T-ALL with immunophenotype &amp; absence of biallelic deletion of TCR-gamma (ABD) &amp; tested its potential significance as prognostic marker. A total of 106 T-ALL patients, including 75 children &amp; 31 adults (males 93; females 13) were included. Immunophenotyping was done in all cases at diagnosis on bone marrow/peripheral blood samples using CD3, CD7, CD4, CD2, CD45, CD5, CD8, CD1a, CD13, CD33, CD117, HLA-DR, CD34, CD65 &amp; CD11b antibodies. The patients were categorized into immature (pre &amp; pro T-ALL; n=48), cortical (n=47) &amp; mature (n=11) T-ALL based on immunophenotypic features. MEF2C gene expression was quantified by RQ-PCR. ABD was done in 90 patients in which DNA was available by quantitative DNA PCR (Q-PCR) for TCR-gamma rearrangements using the protocol described before1. Minimal residual disease in bone marrow samples at the end of induction therapy was also measured. Kruskal-Wallis test was performed to determine the correlation of MEF2C expression with immunophenotypes &amp; TCR-gamma chain status. Kaplan Meier survival analysis was done to evaluate the significance of MEF2C dysregulation on event free survival &amp; overall survival. We found MEF2C gene to be significantly overexpressed (p=0.013) in patients with immature T-ALL as compared to cortical &amp; mature T-ALL cases. It was significantly overexpressed in early T-cell precursor (ETP-ALL) cases (n=15) versus non ETP-ALL (p=0.03). By qPCR, 29 of 90 patients (32.22%) were classified as ABD, 44 (48.89%) as non-ABD &amp; 17 (18.89%) as indeterminate. MEF2C expression was higher in ABD patients (p=0.011). Minimal residual disease was found more frequently in MEF2C dysregulated patients (p=0.033). On Kaplan Meier survival analysis, event free survival (EFS) was not significantly different between two groups (p=0.33). Overall survival (OS) was worse in MEF2C dysregulated patients as compared to others (p=0.017). ETP-immunophenotype was associated with poorer EFS (p=0.013). ABD did not correlate with EFS &amp; OS. To conclude, MEF2C dysregulation was associated with immature T-immunophenotype, ABD &amp; inferior survival at diagnosis. MEF2C gene expression can be used as potential candidate to risk stratify T-ALL cases. Reference 1. Gutierrez A, Dahlberg SE et al. Absence of biallelic TCRgamma deletion predicts early treatment failure in pediatric T-cell acute lymphoblastic leukemia. J Clin Oncol. 2010;28:3816-23. Citation Format: Anita Chopra, Jay Singh, Deepak Verma, Rajive Kumar, Sameer Bakhshi, Jayanth Kumar, Rachna Seth, Atul Sharma. MEF2C dysregulation is associated with immature T cell immunophenotype, absence of biallelic deletion of TCR-gamma and inferior survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5233.

A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium.

T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.

Increased frequency of CD45 negative T cells (T zone cells) in older Golden retriever dogs.

T zone lymphoma (TZL) is characterized by the clonal expansion of T cells lacking expression of the pan-leukocyte antigen CD45 (TZ cells). A strong breed predisposition is observed in Golden retrievers. This study aimed to confirm aberrant CD45 mRNA expression and determine if Golden retrievers without clinical lymphoma have an increased frequency of circulating TZ cells. Gene expression analysis on confirmed TZL cases showed a significant decrease in CD45 expression compared to normal dogs. Peripheral blood samples from senior dogs, 242 Golden retrievers and 42 non-Golden retrievers, without evidence of lymphoproliferative disease were assessed for the presence of TZ cells by flow cytometry. Thirty-one percent of Golden retrievers had TZ cells compared to 14% of non-Golden retrievers. Thirty-four percent of Golden retrievers with TZ cells had a clonal T cell receptor gamma (TRG) gene rearrangement. Interestingly, 20% of Golden retrievers without TZ cells also had a clonal TRG rearrangement. Golden retrievers may have an increased risk of TZL due to an increased frequency of TZ cells.

A Recurrent T-Cell Receptor γ Rearrangement of the Vγ9-JγP Type Represents the Major Clone of γδ T-Cells in Normal Individuals and Is Highly Suppressed in Lymphoproliferative Disorders

INTRODUCTIONγδ T-cells expressing T-cell receptor (TCR) type Vγ9Vδ2 are well-established mediators of anti-tumor immunity and have demonstrable HLA-independent cytotoxic activity against both B- and T-cell non-Hodgkin lymphoma cells. Initial clinical trials using ex vivo expanded, adoptively transferred γδ T-cells to enhance tumor immunity have shown some promise but overall results have been lackluster. This indicates that wholesale polyclonal expansion prior to transfer is not likely to be effective. Thus more refined approaches are necessary. This requires a better understanding of their TCR repertoire. We recently developed a next generation sequencing (NGS)-based method for evaluating the spectrum T-cell receptor gamma (TRG) gene rearrangements present in clinical samples. In an effort to better understand the TCR repertoire of γδ T-cells we used this strategy to evaluate a series of samples from normal individuals and from individuals with B and T-cell lymphoproliferative disorders (LPDs).METHODSDNA was isolated from samples from 11 normal individuals (all peripheral blood, PB), 11 patients with a T-cell LPD (6 PB, 3 bone marrow; BM, 2 FFPE tissues), and 5 patients with a B-cell LPD (Hairy cell leukemia; HCL, 4 PB, 1 BM). γδ T-cells were sorted from the PB of 4 of the healthy donors by FACS. TRG rearrangements were PCR amplified using consensus primers and NGS libraries were prepared and sequenced on the Ion Torrent PGM platform. The data was analyzed as follows. NGS typically yielded up to 400,000 sequencing reads which were grouped by identical V, J, and CDR3 sequences into unique rearrangements (typically 15-30,000). The prevalence of a particular TRG rearrangement (or CDR3 sequence) was determined by the number of individual NGS reads with this unique sequence per the entire data set (percent of total reads). All rearrangements were then ranked by their prevalence.RESULTSWe sequenced the TRG repertoire of isolated γδ T-cells from the peripheral blood of normal individuals (n=4). We found that a recurrent Vγ9-JγP rearrangement with the CDR3 sequence CALWEVQELGKKIKVF was always (4 of 4 samples) the most prevalent rearrangement in normal γδ T-cells (3.2-11.7-fold more prevalent than the second most common rearrangement; representing 4-11.9% of total reads). Similarly, analysis of a larger set of unsorted normal peripheral blood samples demonstrated high prevalence of the same canonical CDR3 in 5 out of 7 samples, confirming that it is very common in most individuals relative to all TRG rearrangements (among the top 10 most prevalent CDR3s in 4 of 5 samples). We also sequenced the TRG repertoire in 11 samples from patients demonstrating evidence of involvement by a T-cell LPD. Unexpectedly, all Vγ9-JγP type rearrangements were strongly suppressed in these samples including the one with the canonical CDR3. The canonical rearrangement (present in 6 /11 cases), or the most abundant Vγ9-JγP rearrangement when the canonical rearrangement was absent (absent in 5/11 cases), represented on average 0.036 ±0.024 % of all NGS reads in the samples from patients with T-cell LPDs compared to 0.72 ±0.72 % of total NGS reads in the normal controls. This represents on average a 19.9 fold reduction in the T-cell LPD samples. The median rank by abundance of the top Vγ9-JγP rearrangement dropped from 9th (normals) to 291st (T-cell LPD cases) indicating that the suppression was not simply a consequence of the presence of an abundant malignant T-cell clone in the data. The overall distribution of TRG V-segment usage in the normal and neoplastic samples was comparable (Vγ9: 9.9 ±0.55 % and 7.2 ±2.79 %, respectively). In 2 samples from patients with HCL, Vγ9-JγP rearrangements were reduced to a similar extent to that seen in the T-cell LPD cases (9.5-fold reduced; average 0.06% of NGS reads; average rank order 451st). In the other 3 cases of HCL the findings were very similar to those seen in the normal samples.CONCLUSIONSWe identified a recurrent Vγ9-JγP rearrangement by NGS representing the most abundant CDR3 in sorted γδ T-cells from normal individuals. This population, along with other clones with Vγ9 rearrangements, appeared specifically suppressed in all samples from patients with T-cell LPDs and in 2 or 5 samples from patients with B-cell LPDs (HCL), perhaps indicating a role in the disease process. Additional samples from a wider range of T- and B-cell LPDs are being analyzed. DisclosuresNo relevant conflicts of interest to declare.

Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells.

The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.

Strategies for Relapsed Peripheral T-Cell Lymphoma: The Tail That Wags the Curve

A 69-year-old woman was referred for further evaluation and management of relapsed angioimmunoblastic T-cell lymphoma. At diagnosis, she received six cycles of dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) and achieved a complete response (CR). Her first surveillance computed tomography scan 3 months later demonstrated enlarging cervical lymphadenopathy. A lymph node excision confirmed relapsed angioimmunoblastic T-cell lymphoma with atypical lymphocytes expressing CD3, CD4, CD10, PD-1, and EBER, with loss of CD5 (Fig 1). A clonal T-cell receptor beta and gamma rearrangement by polymerase chain reaction was identical to that in her initial diagnostic biopsy. At our initial consultation, options for standard as well as investigational therapies were discussed, and HLA typing was initiated. The patient was enrolled onto an investigational phase II study; however, she developed progressive disease after two cycles. She was then treated with romidepsin 14 mg/m(2) administered intravenously for 3 consecutive weeks with 1 week off. After two cycles, she achieved a partial response, and after four additional cycles, she maintained her response without further improvement. We discussed additional treatment options.

A Rare Case of Colorectal Peripheral T-Cell Lymphoma Associated with Mycosis Fungoides

Purpose: ‘Mycosis Fungoides' (MF) is an indolent T cell lymphoma, involving primarily skin with rare extracutaneous, or visceral involvement occurring in advanced stages of the disease. Gastrointestinal system involvement is very rare and it is associated with significant morbidity and mortality secondary to various complications. We are describing a case of “Peripheral T-Cell Lymphoma” involving the rectum and the colon diagnosed on a screening colonoscopy in a patient with a history of Mycosis Fungoides. A 66-year-old AA man with h/o of HTN, dyslipidemia, and mycosis fungoides was referred by his dermatologist for a screening colonoscopy. He denied any symptoms including weight loss, rectal bleeding, melena, hematemesis or altered bowel movements. His physical exam was notable for diffuse hypopigmented and hyperpigmented patches over the skin, primarily hands and feet. There was no evidence of splenomegaly or lymphadenopathy. He was lost to follow up for five years after a decade of stable disease. He was started on clobetasol topical ointment and narrow band UVB therapy. Colonoscopy showed a large ulcerated mass with greenish black exudate occupying 50-74% of the circumference of the rectum, extending 5cm from entry site up to 17cm. The biopsy showed colonic mucosa diffusely infiltrated by large atypical lymphoid cells with varied size, hyperchromatic nuclei and small nucleoli. Flow cytometry showed abnormal clonal T cell proliferation and stained positive for CD3, CD4, CD5 and rarely for CD8 suggestive of peripheral T cell lymphoma. A molecular study for TCR Gamma rearrangement performed on the previous lymph node biopsy and the current rectal specimen showed identical clonal peaks indicating that the process in the rectum likely represents transformation of the patient's cutaneous T cell lymphoma. A month later, he was admitted to the hospital with severe pain and swelling in his right lower extremity. He was found to be having a diffuse systemic involvement of T cell lymphoma of plural fluid, right thigh, inguinal lymphnodes, and CNS. His prognosis was assessed to be very poor and he was sent to hospice care based on his wishes for comfort care.Figure: No Caption available.

Phenomenon of Oligoclonality in Adult Patients with Acute Lymphoblastic Leukemia

Abstract 4895 Introduction:Minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) can be analyzed by flow cytometry, fluorescent in situ hybridization (FISH) if there are chromosomal abnormalities, polymerase chain reaction (PCR) for chimerical transcripts and immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRG) genes rearrangements. IgH and TCRG rearrangements are the for monitoring of MRD, because it is possible to find specific clonal tumor marker in 80–90% patients. According to the data of different research groups phenomenon of oligoclonality (more than one clonal rearrangement) is identified in 16–80% cases depending on how much molecular markers are used (Scrideli CA, 2001; Gameiro P, 2002; Thorsten Raff, 2006; Csinady E, 2009; Katsibardi K, 2011). Methods and materials:We studied bone marrow samples from 59 patients with new diagnosed B-lineage ALL (43 patients), T-lineage ALL (15 patients) and one patient with bilinear B-T-ALL. Ph-positive ALL was diagnosed in 6 patients, t(4;11) – in one patient. Patients were treated by standard ALL-protocols based on 8 weeks induction remission, 3–5 consolidation courses and maintenance treatment during 2 years. Patients with Ph-positive B-ALL were treated by the same chemotherapy with imanitinib and one patient with mature B-ALL was treated by NHL-BFM-90 chemotherapy. Detection of IgH and TCRG genes rearrangements were carried out by polymerase chain reaction using standard family-specific primers. Synthesis of patient specific primers was based on defined nucleotide sequence of clonal PCR-product. Monitoring MRD was realized by nested PCR using patient specific primers. Results:IgH and/or TCRG genes rearrangements were detected in 50 of 59 patients (84,7%). IgH complete rearrangements were found in 31of 43 patients (72,1%) with B-lineage ALL, IgH incomplete rearrangements were detected in 12 of 43 patient (27,9%) with B-lineage ALL and in one of 15 patient with T-lineage ALL. TCRG rearrangements were found in 13 of 15 patients (86,7%) with T-lineage ALL and in 24 of 43 patients (55,8%) with B-lineage ALL. Seventeen (34%) of 50 patients had one clonal marker. So, phenomenon of oligoclonality was revealed in 33 patients (66%). Two clonal rearrangements were detected in 23 cases, 3 rearrangements were detected in 7 cases and 3 patients had 4 rearrangements. In 3 of 27 patients, in whom all markers were followed, monitoring of MRD showed different pattern of tumor subclones elimination. Monitoring of MRD in patients who had also chromosomal abnormalities such as t(9;22), t(4;11) was carried out by FISH and PCR in Real-time. In one case the patient had 3 IgH rearrangements and chromosomal abnormality t(4;11) at the diagnosis. Later at various stages of chemotherapy we observed a different clearance of IgH rearrangements, however, when cytogenetic remission was achieved and MLL-AF4 expression was absent there were two of three IgH markers. This fact confirms the presence of several tumor subclones, their different clearance during the treatment and suggests persistence of tumor in this patient. Conclusion:In ALL patients with several subclones MRD monitoring should carried out with all markers, as we proved different subclone clearance suggesting different cells sensitivity to cytostatic drugs. Disclosures:No relevant conflicts of interest to declare.

Residual anogenital lichen sclerosus after cancer surgery has a high risk for recurrence: A clinicopathological study of 75 women

ObjectivesDespite the strong association of lichen sclerosus (LS) and vulvar squamous cell carcinoma (SCC), the role of LS as precancerous lesion is unclear and the risk for recurrent SCC in residual LS after surgery for a LS-associated SCC is unknown. MethodsRecurrences in residual vulvar LS after complete resection of a LS-associated SCC were analyzed in 75 women. Primary SCC, recurrences and 19 biopsies obtained 1–6months before recurrent SCC were evaluated histologically, and for presence of HPV and monoclonally rearranged T-cell receptor gamma locus (mTRG@). Results40/75 patients (53%; primary SCC 25pT2, 9pT1b, 6pT1a) had no recurrence for 64months (range 10–176months), but 35/75 women (47%; primary SCC 1pT3, 18pT2, 13pT1b, 3pT1a) developed recurrences after 42months (range 3–156months). Twenty-five women had 1 recurrence: 13SCC within 18months, 1SCC after 26months, 10SCC and 1 differentiated vulvar intraepithelial neoplasia (d-VIN) after 74months (range 52–136months). Ten patients suffered multiple recurrences: 3 women had 2 recurrent d-VIN, 7 patients had multiple successive de-novo SCC with lymphocytes with mTRG@ in 6 patients. Wider resections correlated with no/late recurrences. Nineteen HPV-negative biopsies before diagnosis of recurrent SCC revealed 4 classical d-VIN and 15 verrucous, atrophic or flat intraepithelial proliferations different from d-VIN. ConclusionWith a 50% recurrence rate after cancer surgery, residual anogenital LS has a high risk for de-novo cancer. Extent of resection of LS-affected skin and activity of residual LS with lymphocytes with mTRG@ are important criteria for recurrences, which develop rapidly through a variety of HPV-negative intraepithelial lesions.

Development of XMRV producing B Cell lines from lymphomas from patients with Chronic Fatigue Syndrome

Previous studies have shown that CFS patients have an increased incidence of lympho-proliferative malignancy compared to the normal population [1]. While the incidence rate of non-Hodgkin’s lymphoma in the United States is 0.02%, nearly 5% of the CFS patients developed the disease. Additionally, development of cancer coincides with an outgrowth of gamma delta T cells with specific clonal T-cell receptor gamma rearrangements. We hypothesized that infection with XMRV and/or other viruses can trigger a dysregulated immune response which favors the development of B-cell lymphoma. In a study of 300 CFS patients, 13 developed lymphoproliferative disorders. Of those tested, 11/11 were positive for XMRV and 9/9 positive for clonal TCR gamma rearrangements. Spontaneous development of four B cells lines occurred during culture of cells from CFS patients. Three developed from B cells isolated from the peripheral blood (two of whom had B cell lymphoma) and one from a bone marrow biopsy. For all four lines, the B cells have a mature CD20+, CD23+ phenotype and produce infectious XMRV at a titer of >106 infectious units/ml. Virus production occurred despite extensive hypermutation of the proviruses in these cells by APOBEC3G.. Therefore XMRV infection may accelerate the development of B cell malignancies by either indirect chronic stimulation of the immune system and/or by direct infection of the B-cell lineage . Since viral load in peripheral blood is low, these data suggest that B cells in tissues such as spleen and lymph nodes could be an in vivo reservoir for XMRV.

Innovative technology for cancer risk analysis

After completion of the Human Genome Project, analysis of genetic and genomic variations in different pathological states became possible. The capillary system based on Sanger methods is still very expensive in terms of time, cost and professionalism required. For this reason, the National Human Genome Institute proposed an 'advanced sequencing technology development' project with the aim of sequencing a genome in 1 day for $1000. Three validated platforms are commercially available and single molecule sequencing methods have been recently introduced, which are not only competitive in time and costs, but display greater accuracy than 'past generation' sequencing. Next generation technology allows, in a single experiment, the identification of copy number variation and large rearrangements, or detection of fusion transcripts analysis thus permitting the evaluation of cancer risk at multiple levels (genomic, transcriptomic, proteomic, epigenetic).

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

Abstract Background: T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Results: Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Conclusion: Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

An aggressive systemic juvenile xanthogranuloma clonally related to a preceding T‐cell acute lymphoblastic leukemia

Juvenile xanthogranuloma (JXG) is a disorder of disputed origin thought to be related to the dermal/interstitial macrophage. A 5-year-old female presented with an aggressive systemic JXG that developed 5 months after the diagnosis of T-cell acute lymphoblastic leukemia (T-ALL). Examination of the T-cell receptor gamma (TCR-γ) rearrangement in T-ALL blasts, JXG infiltrated lymph node biopsies and micro-dissected JXG histiocytes revealed an identical bi-allelic TCR-γ rearrangement in all samples, thus providing evidence for a clonal relationship between T-ALL and JXG in this case.

Clinical significance of clonality and Epstein‐Barr virus infection in adult patients with hemophagocytic lymphohistiocytosis

We assessed the clinical significance of T or B cell clonality and Epstein-Barr virus (EBV) infection in adult patients with hemophagocytic lymphohistiocytosis (HLH) to identify factors related to prognosis. A total of 30 adult patients with diagnosed HLH were included in the study. In all patients, EBV-DNA in peripheral blood was examined by quantitative real-time polymerase chain reaction and bone marrow cells were examined for clonal rearrangement of T cell receptor gamma(TCRG) and immunoglobulin heavy chain (IGH) genes. TCRG clones were detected in 10 patients (33.3%) and IGH clones were detected in 8 patients (26.7%). We found no correlation between clonality and patient outcome. The patients less than 1,000 copies (mL)21 of EBVDNA showed a significantly higher clinical response (P 5 0.008) and longer overall survival (P 5 0.01) than those with high viral load of EBV-DNA. Our results suggest that TCRG and IGH rearrangement do not have any clinical significance in adult patients with HLH, but that high viral load of EBV-DNA may be a risk factor for poor outcomes. In HLH, high viral load of EBV-DNA should thus suggest a prompt approach with aggressive therapeutic interventions.

Improved Polymerase Chain Reaction–Based Method to Detect Early-Stage Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides) in Formalin-Fixed, Paraffin-Embedded Skin Biopsy Specimens of the Dog

In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRgamma) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRgamma gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRgamma gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRgamma gene, localized approximately 2.3 kb upstream of J5, was identified.