T-cell Receptor Beta Gene Rearrangements Research Articles

Mycosis Fungoides: Histopathology and T Cell Receptor Gene Rearrangements in Indian Patients

Introduction: Mycosis fungoides (MF) is the commonest cutaneous T cell lymphoma. MF presents in three stages the patch, plaque, and tumor. The distinction of patch-stage MF from inflammatory dermatoses is a diagnostic challenge. Need for the Study: MF shows T cell receptor gamma and beta gene rearrangements (TCRG and TCRB), detectable by polymerase chain reaction (PCR). Data about the clonality of infiltrates in our population and their utility in diagnosis is lacking. Materials and Methods: Skin biopsies from 50 patients with mycosis fungoides from January 2007 to July 2015 were studied for microscopic features. TCR gamma and beta gene rearrangement assays were performed on 14 patients where formalin fixed paraffin embedded (FFPE) was available. DNA was extracted using the phenol-chloroform method. PCR was performed using primers from Invivoscribe Identiclone assay. Clonality was analyzed using Agarose gel electrophoresis. Results: Of the 50 cases, 37 were patch, nine were plaque, and four were tumor stage. All cases showed disproportionate lymphocyte epidermotropism, 38 showed lymphocyte atypia, and 33 showed adnexotropism (11 eccrotropism and 22 folliculotropism). Clonality was assessed in 14 cases. Of these, 3/9 patch stages were monoclonal (1/3 monoclonal for beta, and 2/3 monoclonal for gamma and beta). 3/3 plaque stage MF was monoclonal (1/3 for gamma, 2/3 for both), and 1/2 tumor stage MF was monoclonal for gamma and beta receptors. Conclusion: The patch stage was the most common presentation. TCR gene rearrangements were noted in 50% of MF, with TCR gamma rearrangements being more common. Plaque and tumor stages yielded higher positive rates.

PCR GeneScan Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

OA14 Subcutaneous panniculitis-like T-cell lymphoma: a mimic of lupus erythematous panniculitis

Abstract Background/Aims Lupus erythematosus panniculitis (LEP) is a rare variant of cutaneous lupus erythematosus characterised by tender, erythematous subcutaneous nodules or plaques on the face, trunk and proximal extremities. Without treatment, profound lipoatrophy frequently occurs, which often has a devastating psychosocial impact. Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of cutaneous T-cell lymphoma, which may appear in a similar manner. SPTCL initially preferentially infiltrates the subcutaneous adipose tissue and symptoms vary widely depending on the stage of the disease. Early-stage disease presents as painless self-resolving subcutaneous nodules on limbs and trunk. Methods We present the case of a young woman who presented with progressively developing, widespread painful cutaneous lesions, pyrexia of unknown origin and general malaise for one month. She had a history of hurthle cell neoplasm and a strong family history of malignancy. Initial examination revealed 1-2 cm firm, skin-coloured subcutaneous nodules over her lower back, chest and thigh, with groin and axillary lymphadenopathy. Further lesions progressively developed over a three-month period, with lesions turning increasingly erythematous and with associated ulceration. Of note, a significant periorbital lesion appeared, associated with an ipsilateral facial palsy. Results Initial bloods revealed an elevated LDH with cholestatic liver function tests. PET-CT imaging showed an enlarged axillary node with extensive pathology throughout the subcutaneous fat of unknown aetiology. Due to the family history, underlying haematological malignancy and atypical Sweet’s were considered for this unusual presentation. Seropositivity for anti-Ro and ENA, alongside raised angiotensin converting enzyme, broadened the differentials to include LEP, mixed atypical connective tissue disease and cutaneous sarcoid. Notably, complement, myositis panel and creatine kinase were normal. Lupus vulgaris was considered due to high local prevalence and excluded with T-SPOT testing. Histopathological analysis of lesional skin from the upper arm found predominantly subcutaneous infiltrates of atypical lymphoid cells with irregular hyperchromatic nuclei and rimming of individual fat cells. T-cell clonality analysis detected clonal T-cell receptor beta (V-J and D-J) gene rearrangements consistent with SPTCL. Her orbital and cutaneous lesions responded well to six cycles of CHOEP (cyclophosphamide, hydroxydaunorubicin, oncovin, etoposide, prednisone) and the patient returned to functional baseline. Conclusion This case demonstrates the diagnostic difficulties when managing a patient with relapsing remitting panniculitis. Whilst the initial presentation of SPTCL here was classical, with widespread multifocal subcutaneous nodules, the appearance of facial symptoms in SPTCL is rare. Diagnostic uncertainty is frequent in both LEP and SPTCL due to the initial non-specific cutaneous presentation. This case emphasises the importance of clinicopathological correlation for accurate diagnosis. Interestingly, up to 20% of SPTCL patients have concomitant autoimmune diseases, and there is evidence suggesting a potential association between LEP and subcutaneous SPTCL beyond chance, leading some researchers to propose that these two entities exist along a spectrum. Disclosure D. Li: None. L. Spencer: None.

Uncommon Variant of CD4+ T-cell Large Granular Lymphocytic Leukemia

Abstract Introduction/Objective T-cell large granular lymphocytic leukemia (T-LGL) is a T-cell lymphoma with persistently increased large granular lymphocytes in peripheral blood. The majority of T-LGL is associated with CD8+ T-cells. However, the CD4+ variant is uncommonly seen. Methods/Case Report The patient is a 49-year-old female with a history of diabetes mellites and anemia who presented with shortness of breath, lower extremities edema, and fatigue. Physical examination showed a significantly distended abdomen. CBC results showed hemoglobin 6.9 g/dL, WBC 18.2 × 109/L with 88% lymphocytes, and PLT 190,000. The peripheral blood smear presented lymphocytosis with LGL cells, microcytic anemia, and increased target cells. Bone marrow biopsy showed lymphoid infiltrate as an intravascular pattern confirmed by immunochemical stains. A complete workup including flow cytometric, cytogenetics, and molecular studies was performed on the bone marrow. The lymphoma phenotype by flow shows positivity for CD2, CD3, CD4, CD5 (bright), CD7 (partial/dim), CD56, CD57 (majority), TCRαβ. T-cell gene rearrangement was detected and was further confirmed by PCR with the detection of clonal TCR beta gene rearrangements on molecular genetics analysis. However, STAT3 was not detected. The patient was diagnosed with CD4+ T-LGL leukemia. Results (if a Case Study enter NA) NA. Conclusion CD4+CD8- immunophenotype is an uncommon variant of T-LGL, which has a high incidence of activating STAT5B mutation, while CD8+CD4- T-LGLL tends to have STAT3 mutation. Clinically, those two subtypes behave similarly. Flow cytometry is the golden method to confirm the diagnosis. More research is needed to clarify the essence of CD4+ T-LGLL.

Peripheral blood T-cell receptor repertoire profiling of advanced non-small cell lung cancer patients receiving PD-1/PD-L1 treatment.

e21004 Background: Whether peripheral blood T cell receptor (TCR) repertoire profiling can serve as a biomarker to predict clinical benefit from anti-PD-1/PD-L1 checkpoint immunotherapy is not well understood. Moreover, it is not known which methods for TCR repertoire analysis are most clinically meaningful. To address this, we have performed TCR repertoire analysis of patients with advanced/metastatic non-small cell lung cancer (NSCLC) receiving PD-1/PD-L1 treatment. Methods: We analyzed 29 patients receiving PD-1/PD-L1 monotherapy or combination therapy in any line of treatment, excluding patients with EGFR mutations or Alk alterations. Genomic DNA was extracted from peripheral blood examples, and CDR3 regions of the rearranged TCR beta genes were amplified and sequenced using the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). Libraries were sequenced using Illumina HiSeq 2500. TCR clonality, diversity, evenness, and the percentage of high-frequency clones (> 0.1% of the repertoire) were calculated at pre-treatment and post-treatment (1 – 3 months) timepoints. Morisita’s overlap index was calculated for 25 paired pre- and post-treatment samples. Metrics were compared in patients with and without durable clinical response at 6 months using the Mann-Whitney test. Results: There were no statistically significant differences in TCR repertoire clonality, diversity, evenness, or high-frequency clones between patients with and without durable clinical benefit, either at pre-treatment or post-treatment time points (p > 0.05). Conclusions: TCR repertoire metrics including clonality, diversity, evenness, percentage of high-frequency clones, and Morisita’s overlap index do not predict immunotherapy responses in this cohort of advanced/metastatic NSCLC patients receiving PD-1/PD-L1 monotherapy or combination therapy. Limitations of this study include sample size and heterogeneity of the patient cohort. This highlights the need for advanced TCR repertoire metrics, for example, considering shared TCR specificities and clonal identity. In ongoing work, we are defining TCR specificity groups and TCR clones in NSCLC as biomarkers of immunotherapy responsiveness.

T-Cell Monoclonality As Assessed By TCR-Sequencing: A Potential Biomarker of Response in Patients with MDS with Autoimmune Cytopenias Treated with IVIG

Background: We have previously reported 75% of patients (pts) achieved clinical responses to treatment with intravenous immunoglobulin (IVIG) in pts with myelodysplastic syndromes (MDS) that exhibit autoimmune cytopenias in the context of T-cell clonality. T-cell clonality was previously determined via peripheral blood (PB) gel-based testing of T-cell receptor (TCR) beta (TRB) and/or gamma (TRG) gene rearrangements or flow cytometry. IVIG was shown to be variably effective in normalizing hemolytic parameters and cytopenias (Feld et al, Blood 2019). Since next generation sequencing of TRG and TRB (TCR-seq) offers better resolution as to the identity of T-cell clones, we reassessed T-cell clonality in a subset of our cohort and longitudinally evaluated the persistence of clones with IVIG treatment. Methods: Among 27 pts who were previously treated with IVIG +/- steroids, we processed the PB of 9 pts who had serial samples of leftover DNA available for TCR-Seq analysis (7 had samples available prior to treatment). Clinical responses were per MDS IWG 2006 criteria, whereas hemolytic responses were established by either a complete normalization or a mild response if there was >50% improvement in hemolytic parameters from baseline. TCR-Seq was performed on 100ng DNA using the Lymphotrack® TRG and TRB Assays on Illumina® MiSeq platform according to manufacturer's instructions (Invivoscribe, Inc.). Using the LymphoTrackMiseq2.4.3 software T-cell clonality was interpreted as monoclonal (MC), oligoclonal, small clone(s) of uncertain significance (SCUS) or polyclonal (PC), based on percent total merged clonal reads and polyclonal background. Results: Baseline characteristics of our 9 pt cohort included 8 males and 1 female, a median age of 70 years (range 45-82), and a median duration of treatment with IVIG of 23 months. Six pts received steroids in addition to IVIG. Diagnosis included 8 pts with MDS and one with large granular lymphocytic (LGL) leukemia. Of the MDS pts measurable by IPSS criteria, 6 were Int-1 and one was low risk. Expanded lymphocyte populations were detected in 7 pts by PB flow cytometry and all pts exhibited T-cell clonality by gel-based assessment. All were TRB positive (+) and 7 were TRG+ by PCR. In 3 pts, 4 serial samples were available for TCR-seq; 4 pts had 3 samples available and 2 had only 2 serial samples. Samples were collected over a period of 5 months up to 4-5 years. With the better resolution afforded by TCR-Seq, 3 pts exhibited only PC populations of T-cells for TRG and TRB at all time points, and 1 pt exhibited SCUS in 1 of 3 samplings for TRG. Of the four pts, only one had an erythroid response; importantly, this sample was tested after treatment. One pt had a normalization in their bilirubin, otherwise only mild responses in hemolytic parameters were noted in the remaining 3 PC pts. Median duration of treatment was 1.5 months. Five pts had a MC TRB and/or TRG population by TCR-Seq. Two pts had MC TRG, two had MC TRG & TRB, and one had MC TRB populations. Four of the 5 pts had hematologic improvement (HI): 1 had an erythroid and platelet response, 2 had an erythroid response, and 1 had a platelet response. More significant hemolytic responses were also observed in 3/5 pts, with a mild response in the 2 remaining pts. Only 1 TCR-Seq+ pt had no HI, and this pt had a monoclonal population detected by TCR-Seq after finishing IVIG treatment. Median duration of treatment was 45 months. For all these cases, the same clonal sequences were found across all samples up to 4-5 years. Of 4 evaluable patients, 3 had reductions in their clonal read percentage burden over their treatment period. Conclusions: TCR-Seq of the TRG and TRB genes affords better resolution of T-cell clones and permits tracking of clones over time. On a further subgroup analysis of 9 pts from our cohort of IVIG-treated pts, we found that pts who exhibited monoclonality as established by TCR-Seq were more likely to respond to IVIG, with more significant erythroid and hemolytic responses, associated with a consequential (45+ months) longer treatment duration. Thus, TCR-Seq appears to be a superior option to PCR-based TCR gene rearrangement assays to reliably identify T cell clones in pts with MDS, and may be a useful biomarker to prospectively identify a subset of pts that can respond to IVIG. Further studies are ongoing to test this hypothesis. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Medimmune: Research Funding; Celgene: Research Funding.

Leukemia Cutis in a Patient with Rare Indolent T-Prolymphocytic Leukemia - A Case Report and Review of the Literature

Abstract Introduction/Objective T-cell prolymphocytic leukemia (T-PLL) is rare, comprising 2% of mature lymphocytic leukemias in adults. Clinical course is usually aggressive, however, 20-30% patients can have an indolent early phase. Most patients present with extensive disease with peripheral blood lymphocytosis, hepatosplenomegaly, and lymphadenopathy. T-PLL has a higher incidence in patients with ataxia-telangiectasia. Methods We present a case of a 67-year old man with history of somatic mutation of ataxia-telangiectasia gene (ATM) and multiple malignancies, including melanoma, skin and laryngeal squamous cell carcinomas, adrenal adenoma and spindle cell lung tumor (diagnosed on biopsy), who developed leukemia cutis during an indolent phase of T-PLL. Results Initial diagnosis of T-PLL was on routine complete blood count when patient had mild atypical lymphocytosis (5.5 x 109/L). Flow-cytometry immunophenotyping detected CD4 + 8 positive mature T-cells with no aberrant loss of T- cell antigens, and molecular studies confirmed clonal T-cell receptor beta and gamma gene rearrangements. T-PLL associated leukemia cutis was diagnosed on a leg skin nodule biopsy 10 months later. Subsequent bone marrow biopsy demonstrated low level involvement by T-PLL. The patient’s T-PLL followed an indolent trajectory for three years and treatment was withheld until symptomatic presentation. Rapid disease progression was heralded with emergence of widespread T-PLL leukemia cutis, rapidly increasing lymphocytosis, extensive lymphadenopathy, splenomegaly and increasing LDH levels. Conclusion Skin involvement is reportedly identified in 20% to 50% of T-PLL cases in some series. Skin manifestations can be heterogenous with a broad clinical differential. T-PLL associated leukemia cutis may not be recognized owing to the overall low incidence and in skin lesions appearing during an undiagnosed indolent early phase of T-PLL.We highlight the importance of accurate evaluation of non-specific skin lesions in early detection of T- PLL. Being aware of this diagnosis and early engagement in an interdisciplinary approach may allow for better outcomes of this aggressive malignancy.

Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Introduction All mature T-cell-based lymphoid malignancies harbor identical (clonal) rearrangements of their T cell receptor (TCR) genes (van Dongen et al. Clinica Chimica Acta. 1991, 198, 1-92). The clonality assessment of the rearranged TCR gene is particularly important for the identification, characterization and monitoring of T-cell neoplasms since histology and immunophenotyping alone is not enough to make conclusive diagnosis in all cases. Diagnostic clonality testing is currently based on the parallel analysis of the rearranged TCR-gamma and TCR-beta chain genes. This is done using a multiplex PCR developed within the European BIOMED-2/EuroClonality consortium (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis of the resultant PCR products. Although, this method is considered the "gold standard", which is established worldwide in many molecular diagnostic laboratories, the resolution of this approach is limited, especially in cases with low percentage of clonally rearranged TCRs and a high mixture of non-clonally rearranged T-cells. Next generation sequencing (NGS) is a powerful tool to provide resolution at single gene level. This also holds true for NGS assays, which are able to detect all unique TCR rearrangements in a given sample with very high resolution and sensitivity. This approach can be employed for precise assessment of the immune repertoire, minimal residue disease and T-cell clonality. Detailed insights into the clonotypes provides a great potential for early diagnosis of T-cell neoplasms, and the identification of individual clones is essential for monitoring of the disease. Methods We investigated 19 formalin-fixed paraffin-embedded (FFPE) tissue samples from celiac disease patients (n = 14) and routine diagnostic cases suspicious of lymphoma (n = 5). After DNA extraction, we performed multiplex PCRs using the BIOMED-2 TCR-beta primer sets (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis. For TCR-beta NGS, the Oncomine™ TCR Beta-SR DNA Assay (Thermo Fisher Scientific) was used according to the manufacturer's instructions and sequencing was performed on the IonTorrent S5. Identification of individual clonotypes and bioinformatics analysis of the data was done with the help of the IonReporter software (Thermofisher Scientific). It is worth noting that the 14 celiac disease samples were previously analysed by a different TCR-beta NGS approach (Ritter et al. Gut. 2018, 67, 644-653) and utilized for the comparison of the NGS-data. Results 17 out of 19 cases analysed by TCR-beta multiplex PCR (BioMed-2) followed by capillary electrophoresis and by the Oncomine™ TCR Beta-SR assay displayed a very similar length distribution of the PCR products. This holds true for samples with a clonal appearance and for samples with an oligo-/polyclonal pattern. In two discrepant cases, the Primerset B of the BioMed-2 approach showed a dominant amplification product, which was not as clear by TCR-beta NGS in which both cases displayed an oligoclonal TCR-beta gene rearrangement pattern with a few dominant T-cell populations. Both samples were from celiac disease patients, which mainly present a pronounced oligoclonal TCR-beta gene rearrangement pattern. Strikingly, the NGS data from the Oncomine™ TCR Beta-SR DNA Assay delivered highly comparable results when compared to the sequencing data of Ritter et al. 2018 despite having completely different primer sets and a different NGS platform. Conclusion Our comparison of the conventional multiplex PCR (BioMed-2) and TCR-beta NGS (Oncomine™ TCR Beta-SR DNA Assay) demonstrated a very high concordance (17/19 cases) of the molecular data. The two discordant cases can be explained by an over-interpretation of dominant species BioMed-2 Primerset B amplifications, which often show up in cases with low T-cell content or oligoclonal T-cell counts. These T-cell clonotypes are detectable by TCR-beta NGS only at a low percentage because of combination of all type of rearrangements in one assay. The robustness and reliability of NGS-based TCR-beta clonality testing was demonstrated by comparison of two completely different assays, leading to very similar results for all celiac disease patients. We are thus very confident that NGS-based clonality testing will be the "gold standard" of the future. Disclosures Looney: Thermo Fisher Scientific: Employment. Allen:Thermo Fisher Scientific: Employment.

PCR GeneScan and Heteroduplex Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

An unusual case of hepatosplenic T cell lymphoma in a 59-year-old woman

Hepatosplenic T cell lymphoma (HSTL) is a rare aggressive T cell lymphoma which can be diagnostically challenging. We report a case of a 59-year-old woman presenting with sepsis, hepatosplenomegaly, ascites and multi-organ failure. The liver biopsy revealed active hepatitis and steatosis, additionally, with small numbers of atypical sinusoidal lymphoid cells, occasionally forming short chains, but without sinusoidal expansion, difficult to identify and partly masked by inflammatory changes. The cells showed CD3+, CD2+, CD5–, CD7–, CD4–/CD8–, CD56+, perforin+, TIA1+ and CD30+, suggesting an NK/T cell lymphoma, possibly HSTL, with peripheral T cell lymphoma, NOS, also considered. This prompted a subsequent bone marrow trephine, which contained a morphologically and immunophenotypically similar infiltrate of atypical lymphoid cells. Monoclonal TCR beta and gamma gene rearrangements were identified. Despite chemotherapy with CHOEP, the patient deteriorated rapidly and three months after initial diagnosis, she developed progressive CNS disease and died two weeks later. The liver and bone marrow findings, taken in conjunction with the clinical presentation including hepatosplenomegaly, absence of lymphadenopathy or atypical lymphoid cells in the blood, were considered most in keeping with HSTL. This case highlights the difficulty in diagnosis and appreciation of the neoplastic infiltrate of HSTL on haematoxylin-eosin stained slides, as the neoplastic infiltrate can be inconspicuous and masked by inflammatory changes. A high index of suspicion and low threshold for performing immunohistochemistry is prudent in such cases.

Abstract LB-053: Next-generation sequencing of T-cell receptor-beta gene rearrangements reveals dramatic expansion of T-cell clonotypes after CART-EGFRvIII therapy for glioblastoma

Abstract We are interested in the T cell repertoire in glioblastoma (GBM) patients who have been administered peripherally infused CART-EGFRvIII adoptive T cell therapy for recurrent EGFRvIII-positive GBM. On neuropathologic evaluation following GBM tumor resection, we noticed that several subjects had a robust lymphocytic infiltrate in their tumors following CART-EGFRvIII infusion, which was out of proportion to the level of CART-EGFRvIII genomic sequences observed. We sought to quantify the T cell infiltrate in five subjects whose tumors were resected at different time points following CART infusion (days 6-104). In these five subjects, we analyzed the T cell clonotypic repertoire in the infusion product and in pre- and post-infusion brain tumor specimens by deep sequencing the T cell receptor (TCR) Vβ chain of tumor-infiltrating T cells. Data analysis was performed using ImmunoSeq and exported data were further examined using Graphpad Prism. The infusion products in these patients were highly polyclonal with no clones represented above 1%. We found that in all five of these subjects, the number and frequency of unique TCRVβ clonotypes increased by several thousand (~20-fold) in post-infusion GBM specimens, and only a small portion of these unique TCRs were shared with pre-existing tumor-infiltrating specific T cells (TILs). A consistent finding in these patients was that the clonotype compositional diversity of T cells in the tumor bed post-infusion was considerably larger than the pre-CART vs. infusion product sharing frequency. These data indicate there was a significant increase in the number and clonotypic diversity of tumor-infiltrating T cells following intravenous CART-EGFRvIII infusion, but only a small proportion appears to be derived from the infusion product. Collectively, these results show that CART infusion can trigger accumulation of TILs. Whether or not these TILs respond to GBM neoantigens remains to be investigated. Citation Format: Donald M. O'Rourke, Fang Chen, Zev A. Binder, Irina Kulikovskaya, Minnal Gupta, Jeffrey Finklestein, David E. Ambrose, MacLean P. Nasrallah, Carl H. June, Simon F. Lacey, J. Joseph Melenhorst. Next-generation sequencing of T-cell receptor-beta gene rearrangements reveals dramatic expansion of T-cell clonotypes after CART-EGFRvIII therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-053. doi:10.1158/1538-7445.AM2017-LB-053

Molecular Immunoprofiling the T Cell Repertoire after Rituximab Administration Reveals Frequent Oligoclonality Albeit with Different Patterns Depending on the Clinical Context

In patients with B lymphoid malignancies, depletion of B cells by Rituximab, an anti-CD20 humanized monoclonal antibody, can induce changes in the subset composition, activation and function of T cells. The spectrum of resultant immune-mediated sequelae encompasses organ-specific manifestations (e.g. pneumonitis, gastritis) as well as Rituximab-associated late onset neutropenia (R-LON). Although the pathogenesis of these clinical syndromes is not fully elucidated, evidence suggests that at least a fraction of cases may develop in a setting of expanded cytotoxic T cell populations with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Similar cytotoxic T cell expansions can be observed after Rituximab administration in other clinical settings e.g. allogeneic transplantation (allo-HCT), where selective restriction of the T-cell receptor (TR) gene repertoire is probably driven by multifactorial mechanisms. Here, we sought to obtain more insight into this phenomenon by molecular immunoprofiling of the TR gene repertoire in two groups of patients who received Rituximab: (i) Group A: patients (n=10) with chronic lymphocytic leukemia (CLL) treated with fludarabine-cyclophosphamide-rituximab (FCR); and, Group B: patients (n=14) who underwent allo-HCT for hematologic malignancies and received Rituximab either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. Each patient included in the study received a mean of 6 cycles of Rituximab (range, 1-14). TR repertoire analysis was performed 11-88 (median, 36) and 5-24 months (median, 5) after the first Rituximab administration in Group A and Group B, respectively. TR beta gene rearrangements were PCR amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data were analyzed using the IMGT/V-QUEST tool. A total of 579 productive TRBV-TRBD-TRBJ rearrangements were analysed, 291 for Group A, 288 for Group B (6-91/case, median=20). Among the 46 TRBV functional genes identified, 3 accounted for >25% of cases in both groups: (i) TRBV27 (13% in both Groups A and B); ii) TRBV19-1 (13% in group A, 7% in group B); and, (iii) TRBV6-1 (7% and 6%, respectively). Clusters of identical (>=2) rearrangements corresponding to clonotypes were identified in all patients. Oligoclonality with immunodominant clonotypes (>12% of the repertoire) accounting for over 30% of the analyzed sequences was more frequent in Group A (7/10 cases) versus Group B (5/10 cases); however, larger clonotype expansions were seen in group B. Longitudinal analysis was performed in 3 patients with oligoclonality, 1 from group A and 2 from Group B: in the Group A patient, immunodominant clonotypes disappeared, while both patients in Group B retained the oligoclonal repertoire. Lymphocyte subpopulation analysis by flow cytometry was performed in 6 patients of each group. T-LGL proliferations (defined as CD4/CD8 abnormal ratio and CD3+CD8+CD57+ >30%) were found more often in Group B (3/6 cases in Group A versus 6/6 in Group B). They were related to oligoclonal TR gene repertoire in Group A (3/3) but not in Group B patients (3/6 cases). However, true expansions could be considered only in group B patients, since CD8+ lymphocytes >1.0*109/l were seen in all 6 Group B versus only 1/6 group A cases. Self-limiting R-LON was observed in 8 patients (4 in each group), but no association of oligoclonality to R-LON could be found. In conclusion, we report frequent development of oligoclonal T cell populations after Rituximab treatment in two different clinical contexts. The Groups analyzed differed with respect to the extent of oligoclonality, suggesting that the precise clinical setting determines the amplitude of TR repertoire skewing after Ritximab. Sustained oligoclonal cytotoxic expansions were recognised more often among allo-HCT patients, presenting with a highly restricted TR gene repertoire and likely reflecting strong antigenic stimulation by viruses and/or cGVHD aggravated by T cell imbalances induced by Rituximab. DisclosuresStamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Characterization of Myelodysplastic Syndromes (MDS) with T-Cell Large Granular Lymphocyte Proliferations (LGL)

▪Introduction:MDS include a spectrum of hematopoietic stem cell malignancies characterized by bone marrow failure and dysplastic morphology. LGL is a clonal proliferation of cytotoxic T cells, which manifest as neutropenia, anemia, and thrombocytopenia and is associated with autoimmune disorders. LGL in association with MDS has been previously reported. However, clinicopathological features, prognostic, and predictive factors in those patients diagnosed with both LGL and MDS is not well studied.Methods:We identified patients at Moffitt Cancer Center (MCC) diagnosed with MDS who were previously tested for the presence of LGL clonal populations by peripheral blood flow cytometry at time of first visit. An LGL population was defined by the standard flow cytometry immunophenotype and clonality confirmed by T-cell receptor gamma and beta gene rearrangement.. Next Generation sequencing data was available for 151 patients. Recurrent somatic gene mutations were compared between patients with an LGL clone and those without.Results:Of the 675 patients with MDS tested for LGL in the database, 206 (30.5%) had an LGL clonal population. The mean LGL absolute cell count in the peripheral blood was 335/µL. Table-1 summarizes the baseline characteristics of the two groups.There was no difference in response to azacitidine therapy. Among 50 patients with LGL clone who received azacitidine with available data on response, the rate of hematological improvement or better (HI+) was 38%. The (HI+) was 28% among 105 patients evaluable for response without LGL clone. P .14The median overall survival (OS) was for patients with no LGL clone was 65 months (mo) compared to 46 mo (p .024). The median OS for lower risk MDS patients (low/int-1 by International Prognostic Scoring System [IPSS]) was 68 mo versus 97 mo for those with or without LGL proliferation, respectively (P .005). In higher risk MDS, there was no difference in median OS between those with or without LGL expansion, respectively (20 mo versus 16 mo, p .7). The median OS for patients with very low/ low Revised-Internatinal Prognostic Scoring System (R-IPSS) was 96 mo if LGL proliferation was detected compared to 128 mo if it was not, (p value .016). For intermediate R-IPSS the median OS was 65 mo and 41 mo with or without LGL proliferation (p .16). Finally, for high/very high R-IPSS the median OS was 18 and 16 mo with or without LGL proliferation, (p .84) In cox regression analysis the presence of an LGL clone was independently prognostic for OS after adjusting for age and R-IPSS, Hazard ratio 1.3, p = .05. Somatic gene mutation data were available for 151 patients; there was no statistically significant difference in the distribution of any mutation except IDH-2 (Table-2). The most common somatic mutations observed among patients with LGL clone were SF3B1 19%, TET-2 16%, U2AF1 13%, IDH-2 13%, RUNX-1 13%, and ASXL-1 10%. In patients without an LGL clone the most common somatic mutations were TET-2 26%, ASXL-1 20%, DNMT3A 16%, TP53 13%, SF3B1 12%.Conclusion:An LGL clone is demonstrable in approximately 30% of patients with MDS in association with advancing age. The presence of LGL proliferation was associated with worse OS in lower risk MDS pts. Although the spectrum of somatic gene mutations were similar, the presence of IDH-2 mutation and absence of DNMT3A or TP53 gene mutationscharacterized LGL+ cases. [Display omitted] [Display omitted] DisclosuresRoe:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Seattle Genetics: Speakers Bureau. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy.

Aggressive T-LGL or indolent Hepatosplenic T-cell lymphoma: case with a difficult differential diagnosis

T-cell large granular lymphocytic leukemia (T-LGL) is an indolent lymphoproliferative disorder characterized by persist en t (>6 months ) i nc rease in pe r iphe ra l b lood immunophenotypically distinct, clonal population of large granular lymphocytes, usually between 2–20×10^9/L, without a clearly identifiable cause [1]. Hepatosplenic T-cell lymphoma (HSTL), on the other hand, is a uniformly aggressive disease with a fulminant systemic clinical presentation with massive hepatosplenomegaly, bone marrow involvement, thrombocytopenia and anemia, and B symptoms with survival counted in months (6–16 months) from the time of diagnosis [1]. More indolent variants are not recognized. This is a case with clinical presentation more compatible with T-large granular lymphocytic leukemia (T-LGL) and clinically followed as such, until it manifested clinically aggressively with B symptoms, splenomegaly, lymphadenopathy, and morphologically compatible with HSTL. This case continued to challenge us with the differential of a case of T-LGL with aggressive transformation or a HSTL with indolent presentation. This is a case of a 65-year-old lady, with a 10-year history of Raynaud’s disease, who presented with a mild viral syndrome associated with a slight leucopenia in September of 2007. In 2008, due to persistent cytopenia, a bone marrow biopsy was obtained. Her CBC at that time showed a wbc 1.4 k/uL, with absolute neutropenia, Hg 13gm/dl, and platelets 137 K/uL. There were no B symptoms (fever, weight loss, night sweats). The peripheral smear failed to show an abnormal lymphocyte or large granular lymphocytic population. The bone marrow biopsy showed a small inter-trabecular nodular CD3-positive T-lymphocytic aggregate without significant interstitial T-cell infiltrates. Flow analysis showed an aberrant T-cell phenotype CD3+/CD8dim+/CD7dim+/ CD56dim+/CD2+/CD16+/CD4−/CD5−/CD57− suspicious for a T-cell lymphoproliferative disorder. In 2009, the patient presented with pancytopenia, marked splenomegaly and mild hepatomegaly. The CBC showed wbc 1.7 (Neutrophil 340/ uL), Hg 10.8, and plt 80. The bone marrow was hypercellular marrow (70 %) with decreased granulocytes, increased erythroid precursors, and normal-appearing megakaryocytes. There was an interstitial and moderate intrasinusoidal T-cell infiltrate containing small lymphocytes with round to slight irregular nuclear profiles and ample cytoplasm (HE, CD3+, TIA1+, Granzyme B equivocal negative) Fig. 1 (a–c). Flow analysis showed a similar aberrant phenotype CD3+/ CD8dim+/CD7dim+/ CD56dim+/ CD2+/CD16+/CD4−/ CD5−/CD57− and γδ T-cell receptor-positive. Molecular studies were performed, and TCR beta and gamma gene rearrangements were seen. Cytogenetic studies showed a normal karyotype. FISH studies performed showed no chromosome 7 abnormalities. She was treated with G-CSF for her cytopenias. Unfortunately, this caused a splenic rupture (Fig. 1d). The spleen showed a diffuse red pulp disease with atrophic follicles. In June of 2010, she presented with B symptoms with fevers, night sweats, and a 10-pound weight loss and paraaortic and inguinal lymphadenopathy. There were persistent cytopenias: wbc 2.8, rbc 3.18, plt 204 k. A lymph node biopsy was obtained. The lymph node architecture was effaced by a diffuse population of large transformed lymphocytes (Fig. 1e). By immunostains, they marked as CD3+, CD8+, TIA+ T-cell infiltrate with flow analysis showing a similar phenotype as reported earlier. A peripheral smear reviewed at this time showed numerous large granular lymphocytes (Fig. 1f). She * Prabhjot Kaur prabhjot.kaur@hitchcock.org

T-Prolymphocytic Leukemia Is Sensitive to Polyclonal T Cell-Derived Graft-Versus-Leukemia Effects: Evidence from Minimal Residual Disease Kinetics and TCR Repertoire Diversity Analyses

▪ INTRODUCTIONT-prolymphocytic leukemia (T-PLL) is a rare T cell malignancy with an aggressive clinical course. Preliminary clinical data suggest that allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in a proportion of patients. However, direct evidence that graft-versus-leukemia (GVL) activity is indeed effective in T-PLL is lacking. We sought to investigate GVL in T-PLL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions (immunosuppression tapering, donor lymphocyte infusions (DLI), chronic graft-versus-host disease (cGvHD)), and T cell receptor (TCR) repertoire diversity alterations after alloSCT. METHODSThe study sample consisted of 10 consecutive patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma (TRG) gene rearrangements. Data interpretation followed EuroMRD guidelines. In selected patients, TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS). TRBV-TRBD-TRBJ gene rearrangements were amplified according to BIOMED2 protocol on genomic DNA. PCR products were sequenced on Illumina's MiSeq platform. NGS data were analyzed through a purpose-built bioinformatics immunoprofiler (EuroClonality-NGS consortium). Rearrangements with very similar junctional amino acid sequences and identical TRBV and TRBJ gene usage were defined as clonotypes. RESULTSPatients underwent alloSCT in remission after first-line (8) or salvage (2) alemtuzumab-based therapy. 5 patients were allografted with an unrelated donor, 4 with a related donor, and one received haploidentical alloSCT. Conditioning was fludarabine with cyclophosphamide and/or total body irradiation-based. All patients had a cytological complete response (CR) after alloSCT. 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 81+ and 12+ months post transplant), whilst MRD re-increased in 3 patients after 5-28 months despite ongoing cGvHD in one of them. NGS of the TCR repertoire was performed longitudinally in 3 patients with the longest follow-up. A total of 104 samples (blood (n=91) and BM (n=10) plus 3 donor blood samples) were sequenced. The sample at diagnosis showed one or two major clonotypes in all 3 sequenced T-PLLs, predominantly reflecting a mono- or biallelic leukemic TRB gene rearrangement. Kinetics of this leukemic clonotype followed kinetics of RQ-PCR MRD measurement, demonstrating that NGS can be used to quantify MRD in T cell lymphoma. Immediately after transplantation, the TRB repertoire was heavily skewed in all 3 patients, but recovered over time. Also in all 3 patients, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. This corresponded to the donor clonotype repertoire and disappeared with increasing MRD levels. In each of the samples after alloSCT, several expanded non-leukemic clonotypes were observed at a maximum frequency from 3% to 54% of total TRB sequences. However, during MRD response, novel dominant clonotypes that could explain a clonal GVL effect did not emerge. Figure 1 shows an example of a patient repeatedly showing MRD response to immune interventions. Currently, 5 patients are alive and in cytological CR (4-81 months after alloSCT), translating into a median relapse-free survival of 40 months. CONCLUSIONSThe MRD responses to immune interventions observed here provide the first direct evidence for the efficacy of GVL in T-PLL. However, the GVL effect in T-PLL appears to be often only limited or transient. It also does not seem to be caused by the emergence of novel dominant T cell clones but is rather relying on a poly-/oligoclonal T cell response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.

Aggressive Subcutaneous Panniculitis-Like CD30+ Peripheral T-Cell Lymphoma with Diffuse EBER Expression.

T-cell lineage lymphoma with an intense membranous and paranuclear CD30 expression in the absence of ALK1 raises a differential diagnosis of peripheral T-cell lymphoma (PTCL), NOS and anaplastic large cell lymphoma (ALCL), ALK negative. However, Epstein-Barr virus is consistently negative in ALCL and is not considered an implicating factor in its pathogenesis. We describe a case of T-cell lymphoma showing anaplastic large cell morphology with scattered hallmark cells and a uniform CD30 and Epstein-Barr virus encoded early RNA (EBER) expression that primarily involved the subcutaneous tissue at presentation. On incisional biopsy, the neoplastic cells were positive for CD3, CD2, and CD30 while negative for LCA, CD20, PAX5, CD56, ALK1, and cytotoxic granules. Molecular analysis identified a positive T-cell receptor (beta and gamma) gene rearrangement by PCR. Proliferation index approached 100% and the patient had a rapidly progressive course; the subcutaneous lesions more than doubled in size within couple of weeks with new evidence for widespread systemic involvement. This case emphasizes a rare EBV association with a CD30 positive T-cell lymphoma where the morphologic and immunophenotypic findings are otherwise nondiscriminatory between PTCL, NOS and ALCL, ALK negative.

The Surface Glycoprotein of Feline Leukemia Virus Isolate FeLV-945 Is a Determinant of Altered Pathogenesis in the Presence or Absence of the Unique Viral Long Terminal Repeat

Feline leukemia virus (FeLV) is a naturally transmitted gammaretrovirus that infects domestic cats. FeLV-945, the predominant isolate associated with non-T-cell disease in a natural cohort, is a member of FeLV subgroup A but differs in sequence from the FeLV-A prototype, FeLV-A/61E, in the surface glycoprotein (SU) and long terminal repeat (LTR). Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in pathogenesis indistinguishable from that of FeLV-A/61E, namely, thymic lymphoma of T-cell origin. In contrast, substitution of both FeLV-945 LTR and SU into FeLV-A/61E resulted in multicentric lymphoma of non-T-cell origin. These results implicated the FeLV-945 SU as a determinant of pathogenic spectrum. The present study was undertaken to test the hypothesis that FeLV-945 SU can act in the absence of other unique sequence elements of FeLV-945 to determine the disease spectrum. Substitution of FeLV-A/61E SU with that of FeLV-945 altered the clinical presentation and resulted in tumors that demonstrated expression of CD45R in the presence or absence of CD3. Despite the evident expression of CD45R, a typical B-cell marker, T-cell receptor beta (TCRβ) gene rearrangement indicated a T-cell origin. Tumor cells were detectable in bone marrow and blood at earlier times during the disease process, and the predominant SU genes from proviruses integrated in tumor DNA carried markers of genetic recombination. The findings demonstrate that FeLV-945 SU alters pathogenesis, although incompletely, in the absence of FeLV-945 LTR. Evidence demonstrates that FeLV-945 SU and LTR are required together to fully recapitulate the distinctive non-T-cell disease outcome seen in the natural cohort.

Collision Anaplastic Large Cell Lymphoma (T-Cell/Histiocyte-Rich) and Diffuse Large B Cell Lymphoma: A Pathologic and Clinical Evaluation

A collision tumor of T and B-cell lymphomas occur rarely. In this article we report a collision tumor of anaplastic large cell lymphoma and diffuse large B cell lymphoma in a 46 year old female. The tumor showed predominantly neoplastic anaplastic large cell lymphoma (ALCL) component expressing CD30 and ALK with smaller areas of CD20+ diffuse large B cell lymphoma component. Polymerase chain reaction for T-cell receptor beta and IgH (VDJ) gene rearrangements detected a clonal T cell and a clonal B cell population. The patient developed CSF involvement approximately 3 months after treatment. CSF analysis at this time showed only monoclonal T cells, probably due to clearing of the B cell component by the chemotherapy. To the best of our knowledge this is the first case report of a composite tumor of ALCL (lymphohistiocytic variant) and DLBCL. This case raises issues related to the classification of these composite lymphomas and the treatment on initial presentation and during relapse.

Atraumatic Splenic Rupture: An Unusual Manifestation of Acute HIV Infection

A 27-year-old white male, who had sex with other men, presented to the emergency department with 3 days of left shoulder and abdominal pain. He reported no history of trauma to the abdomen. On abdominal imaging, he was found to have hemoperitoneum from a ruptured spleen; he underwent splenectomy. Causes of atraumatic splenic rupture can be divided into six main categories: infectious, neoplastic, inflammatory, congenital or structural, iatrogenic, and idiopathic. Work-up of the atraumatic splenic rupture revealed that his HIV antibody was newly positive. He had a documented negative HIV antibody 3 weeks prior to the current admission. CD4 cell count, obtained after splenectomy, was 904 cells per microliter and the HIV-1 plasma RNA level was 4657 copies per milliliter. Spleen pathology demonstrated an enlarged spleen with increase in the number of small to intermediate size lymphoid cells in the red pulp, and reactive follicular lymphoid hyperplasia, with numerous secondary lymphoid follicles and reactive germinal centers in the white pulp. T-cell receptor (TCR) gene rearrangement studies demonstrated a positive TCR beta gene rearrangement, without a TCR gamma gene rearrangement, consistent with a clonal CD8(+) T-cell population. The case gives rare insight into what happens in the spleen during acute HIV infection and encourages HIV testing in those presenting with atraumatic splenic rupture. Counseling patients with acute HIV to avoid potential trauma should also be considered.