TCR Rearrangement Research Articles

Absence of Chromosomal Abnormalities Corresponds to Better Survival in Adult Ph-Negative ACUTE Lymphoblastic Leukemia – Results of the Russian ACUTE Lymphoblastic Leukemia (RALL) Study Group.

AbstractAbstract 2572Adult ALL regardless recent advantages in adolescents and young adults is still considered to be a therapeutical problem. So called “a pediatric-like approach” applied in adult ALL is reported to be more reasonable even in the older adults (up to 55 y).RALL has initiated a prospective multicenter trial for adult Ph-neg ALL based on: 1) evaluation of b/m blast clearance after 7 days of Prednisolone (PRD) prephase and its substitution by Dexamethasone (DEXA) if b/m blast count was >25%; 2) continuous 2,5 years treatment schedule with prolonged L-asparaginase (Σ=590.000 IU); 3) evaluation of the impact of the late intensification (2 courses of HD of methotrexate and ARA-C) on MRD clearance. The study is registered on the ClinicalTrials.gov public site; NCT01193933.From Nov, 2008, till June, 2012, 24 centers enrolled 173 pts: median age 28 y (15–55), 71f/101m, 112 (64,7%) = B-lin, 54 (31,2%) = T-lin, 1 pt -undifferentiated AL (0,6%), 6 - uknown phenotype (3,5%), median LDH=848 ME (72–13061), median L=13,5 (0,6–556*109/l). Cytogenetics was evaluable in 58,3% of pts (n=101) and 46,5% of them (n=47) had normal karyotype (NK). Initial risk group was evaluated in 154 pts among whom 46 patients (29,9%) were in the standard risk (SR) group (WBC <30 for B-Lin, <100 for T-Lin, EGIL BII-III, T-III; LDH < 2N, No late CR, t(4;11)-negative), 109 (60,1%) - in the high risk (HR) group (WBC >30 for B-Lin, >100 for T-Lin, EGIL BI, T-I-II-IV; LDH > 2N, No late CR, t(4;11)-positive). 19/173 pts (11%) were not qualified. The analysis was performed in June, 2012. +8 day b/m blast count was reported in 149 pts and b/m blasts less than 25% were detected in 36,2% of pts. The portion of PRD non-responders was statistically different in SR and HR groups: 24/45 (53,3%) and 68/101 (67,3%) (p=0,01), confirming the initial risk groups identification.Induction results were obtained in 150 pts, and CR rate was identical in both risk groups (SR=86,9%; HR=84,1%) with total 12 induction deaths (8,0%) and 6 resistant leukemias (4,0%). With a median follow-up of 12 mo (1–36 mo) death in CR was reported in 9/132 (6,8%) pts. OS at 36 mo was 58,6%, DFS-68,3%.MRD analysis for clonal IgH and TCR rearrangements was carried out in 25 pts. And as in our previous studies (ASH 2006, abstr 2294) the clearance was slow with only 41,6% pts negative for MRD at day +133 (4mo) of the protocol. Two late intensification courses (day +157 = 5 mo) increased MRD negativity only up to 50%. Such slow MRD clearance did not correspond to higher relapse rate so far.Age, WBC, immunophenotype, LDH, risk group, +8 day b/m blast count, time to CR, time without treatment (<>8days), L-asparaginase cessation did not influence survival. OS and DFS differed in pts with NK vs all other abnormalities: 87,4% vs 57,9% (p=0,002) and 88,9% vs 66,5%, respectively (p=0,02).So, our data demonstrated that ALL-2009 protocol provided 58% 3-years overall survival and 68,3% DFS. In adult Ph-neg ALL normal karyotype predicts better survival. The MRD clearance is very slow while on this protocol. Disclosures:No relevant conflicts of interest to declare.

Tle4 Is Critical for Proper B- Cell Differentiation and Maintenance of the Murine Bone Marrow Niche

AbstractAbstract 1202Transducin-like/Enhancer of Split 4 (TLE4), a homolog of the master developmental regulator in Drosophila, Groucho, is a co repressor previously described by our group as the putative tumor suppressor lost in a subset of t(8:21) AML patients also harboring 9q deletion. TLE4 has been found to be of fundamental importance in the repressive function of multiple highly conserved transcriptional complexes, including Hes1, Tcf/Lef, CSL, Runx1-3, and Pax5, all of which have been shown to be intimately connected to normal steady state hematopoiesis and deregulated in malignancy. Owing to its deletion in leukemia and its ability to confer functionality to a variety of signaling networks, our lab sought to investigate its role in hematopoiesis by generating a murine knockout model for Tle4.While normal at birth, within 2–3 weeks of age knockout (KO) animals become runted and eventually moribund by the time of weaning. Gross examination of the spleen and thymus in 3 week old animals reveals significant atrophy of these organs with severe abnormalities in normal tissue architecture. Tle4−/− mice lack clear demarcations between medullary and cortical regions of the thymus, in addition to having a significant increase in apoptosis seen via TUNEL staining. FACS analysis of the thymus shows CD4/CD8 double positive cells are heavily reduced in KOs, with an increase in both single positive mature populations. Additionally, there is a partial, but not complete, block in T cell progenitor development through TCR rearrangement (DN2-DN3 stages). Structural defects are seen in the spleen as well, with a failure in the formation of follicular zones. B cells are significantly reduced with an increase in CD11b+ cells in the spleen, in addition to a shift in the percentages of B1, B2, and marginal zone B cells in some animals. Histological examination of the long bones reveals dramatic abnormalities, both in hematopoietic and bone lineages. Tle4−/− mice exhibit a reduction in calcified bone, a dysplastic and reduced hypertrophic zone, and reabsorption of the trabecular region. These mice also have a gradual clearing of the marrow itself, with 2 week old animals first showing evidence of cytopenia that then progresses to full marrow failure by 3–3.5 weeks of age. The bone marrow exhibits a marked leukocytopenia, with a clear B lymphocytopenia as in the spleen. Furthermore, within the bone marrow, there is an increase in the number of CD4 and decrease in the number of CD8 T cells. ProB staging of developing B cells also trends towards a partial arrest in the transitions from the A through C fractions. When analyzed for progenitor populations, KO mice show severe reductions in the number of common lymphoid progenitors, while myeloid progenitors show no particular shifts in population frequency. Finally, there is a significant decrease in HSCs, defined as lin-Sca1+Ckit++ (LSK).In order to assess whether the hematological phenotype observed in these mice are of a cell intrinsic or extrinsic nature, transplants were performed using embryonic day 13.5 fetal liver and 2 week old bone marrow. Analysis of recipient animals 16 weeks after transplant revealed leukocytopenia with a specific reduction in B cells in the blood, however the T cell and LSK defects seen in donor animals was not observed. Furthermore, when LSK CD34+ Flt3+ or – cells from KO or WT 2 week old animals were plated on OP9 cells over-expressing Deltex1 or GFP, there were no defects in T, B, or CD11b+ cell production.The unique spectrum of phenotypic consequences concomitant to Tle4 deletion argues for a complex yet fundamental role in the maintenance of the hematopoietic niche. As illustrated by the ability to recapitulate some yet not all of the KO hematological aberrations in transplants, it appears that Tle4's involvement in lineage commitment is heterotypic in nature, an unsurprising finding considering its direct involvement in multiple highly conserved signaling pathways. Considering its principal role in those pathways, continued efforts to investigate the role the TLE family of corepressors play in the bone marrow niche may prove vital in reworking our paradigms for both normal and malignant hematopoiesis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinicopathologic Features, Prognosis, and Therapeutic Responses in Patients with Granulomatous Mycosis Fungoides: A Single Center Experience From Memorial Sloan Kettering Cancer Center.

Abstract 2643 Background:Granulomatous mycosis fungoides (GMF) is a variant of MF/cutaneous T-cell lymphoma characterized by a prominent granulomatous infiltrate in addition to the malignant lymphoid infiltrate. The diagnosis of GMF remains challenging due to variable clinical and histopathologic features. The significance of granuloma formation in CTCL is unclear and whether patients with GMF have a distinct prognosis compared to patients with classic type of MF is controversial. Data are scarce and long term results are not available. Objectives:To evaluate the clinical, histopathologic, prognostic significance and therapeutic responses of patients with GMF and to compare results to age and stage-matched patients with classic type of MF. Methods:A single center retrospective case-case study was performed at Memorial Sloan-Kettering Cancer Center of 430 patients with a diagnosis of MF between January 1981 and April 2012. Available histopathology slides were reviewed for the presence of granulomas or histiocytes comprising ≥ 25% of the atypical lymphoid infiltrate. Each identified case was matched with two classic MF cases via age and TNMB stage. For each case meeting criteria, the medical records, photographs, and histopathology slides were reviewed. Results:Twenty-seven patients with GMF were identified representing 6.3% of all MF patients at our center. Patient demographics were similar between the GMF group and case-control classic MF group, with a male-to-female ratio of 2.1:1 and a median age of 57. Most GMF patients (69%) present at early stages (IA-IIA). Patients with GMF had a longer onset of disease prior to diagnosis (median, 4.5 years vs 3.0 years). Skin manifestations of granulomatous MF showed a great variability, but were not distinguishable from those of classic MF. Histological findings showed an atypical predominantly CD4+ lymphocytic infiltrate with either granuloma formation or histiocytes with giant cells. TCR rearrangement was positive in 70% of cases. Features of GMF were present at initial diagnosis in 17 patients. In 10 patients, granulomatous infiltrates appeared at an average of 5.4 years after MF diagnosis. Secondary malignancies developed in 38% of patients with GMF compared to 23% of patients in the case-control classic MF group. Among the GMF patients, there was a trend towards fewer CRs to any treatment (38% vs 54%; p = 0.21); fewer PRs or CRs with topical therapy (57% vs 83%; p = 0.002) and with UV-light therapy (62% vs 90%; p = 0.008). Significantly more GMF patients received systemic therapy (66.7%) compared to classic MF patients (32.7%; p = 0.006). They also required a longer time to achieve best response (median 35 months vs 9 months, p = 0.002) compared to the control group. Disease progression to higher stage was seen in 46% of cases in the GMF group compared to 30% of cases in the classic MF group (p = 0.23). The 5-year and 10-year progression-free survival rates were significantly lower in the GMF group (59% and 33%, respectively) compared to the control group (84% and 56%, respectively; p = 0.02). However, the 5-year and 10-year overall survival rates were similar between GMF (86% and 72%, respectively) and classic MF patients (85% and 85%; p = 0.54). Conclusion:Our study presents the largest series on GMF in the United States. Most patients with GMF show similar clinical features when compared to patients with conventional MF. The clinical features do not reflect the histologic findings of granulomas. More frequent disease progression and poorer response to skin-directed therapies are seen in GMF patients compared to classic MF; however, overall survival is not significantly different. The majority of GMF patients present with granulomatous features at initial diagnosis and in subsequent biopsies suggesting that this entity has a unique relationship with its microenvironment. Disclosures:No relevant conflicts of interest to declare.

Intensified Chemotherapy Regimen for Very High Risk Childhood B-Precursor Acute Lymphoblastic Leukemia (B-ALL): Results From Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05–01

AbstractAbstract 3563 Background:High levels of minimal residual disease (MRD) at the end of 4-weeks of multiagent induction chemotherapy have been shown to be associated with a high risk of subsequent relapse in pediatric patients (pts) with B-ALL. Published reports indicated that pediatric B-ALL pts with high end-induction MRD had event-free survival (EFS) rates < 50% when treated with standard chemotherapy.[Zhou, Blood 2007; Borowitz Blood 2008] Pts with high-risk (HR) cytogenetic abnormalities, such as low hypodiploidy and MLL gene rearrangements (MLL-R) also have a high relapse risk. On DFCI ALL Consortium Protocol 05–01, we piloted an intensified regimen for B-ALL pts with high end-induction MRD and/or HR cytogenetics. Methods:Between 2005–2010, 482 evaluable pts aged 1–18 years with B-ALL were enrolled. Pts were initially classified as standard-risk (SR) or high-risk (HR) based on NCI age/WBC criteria. MRD was prospectively evaluated at the end of the 4-week induction phase via RQ-PCR analysis of IgH or TCR rearrangements. Results were reported as the ratio of copy numbers of target gene:GAPDH; a ratio >0.001 was considered high MRD. Pts with high MRD or HR cytogenetics (hypodiploidy with <45 chromosomes or MLL-R) were reclassified at the end of induction phase as very high risk (VHR), and received 2 additional chemotherapy cycles beginning at week 7 (cycle 1: cyclophosphamide, low-dose cytarabine, 6-MP; cycle 2: high-dose cytarabine, etoposide, dexamethasone, L-asparaginase), followed by the DFCI ALL Consortium HR consolidation phase, including 30 weeks of L-asparaginase and doxorubicin to a cumulative dose of 300 mg/m2. After consolidation, pts received a standard maintenance phase. Total duration of treatment was 25 months. Results:51 pts (11%) were classified as VHR, 25 of whom had been initially classified as SR and 26 as HR. 35 VHR pts had high end-induction MRD as the sole VHR criterion; 16 had HR cytogenetics. 9 pts relapsed (all marrow-involved) and 1 pt developed a secondary AML. There were no deaths in first remission. With median follow-up of 4.4 yrs, the 5-yr EFS (95% confidence interval) for all 51 VHR pts was 76% (60,87)[Figure 1] and 5-yr overall survival was 81% (60,92). The 5-yr EFS was 81% (59,90) for the 35 pts with high MRD. Conclusion:Intensification of chemotherapy (without stem cell transplant) resulted in a relatively favorable EFS in VHR B-ALL pts (defined by the presence of either high end-induction MRD or HR cytogenetics). More pts and longer follow-up will be necessary to confirm these promising results. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

STAT3-Mutations Indicate the Presence of Subclinical Self-Reactive Cytotoxic T Cell Clones in Aplastic Anemia and Myelodysplastic Syndromes

AbstractAbstract 646Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias, but can also occur in the context of myelodysplastic syndrome (MDS). LGL shares certain pathogenetic similarities with aplastic anemia (AA) and some forms of MDS, in which cytopenias are related to immune suppression of normal hematopoiesis. In these conditions, the inhibition of hematopoietic progenitor and stem cells has been described to be mediated by mostly polyclonal cytotoxic T lymphocytes (CTL). Previously, using molecular analysis of TCR VB repertoire in these diseases we have demonstrated oligoclonal skewing clonal of CTL spectrum that was reminiscent (albeit less pronounced) to that seen in LGL. These observations support the theory that these CTL expansions correspond to a cellular reaction against autologous hematopoietic targets. Detection of STAT3 mutations would substantiate the hypothesis that autoimmune reactions can be due to intrisic genetic lesions in autoimmune cells.The recent discovery of recurrent somatic STAT3 mutations appears to be the key molecular lesion promoting clonal outgrowth of autonomous CTL clones in LGL. This finding raised the hypothesis of whether those mutations could be found in other bone marrow failure (BMF) states and whether they could be diagnostically useful and associated with distinct clinical features. Initially, we have directly sequenced STAT3 exons in 120 T-LGL cases and identified 33 mutations in 32 cases (27%). All mutations were located in the domain of STAT3 (residues 585–688) that shares homology with Src homology 2 (SH2) domains. The STAT3 SH2 domain mediates STAT3 dimerization via binding of phosphotyrosine residue Y705. Two mutations, Y640F and D661Y, accounted for 80% of the somatic variations found, enabling the design of a more sensitive ARMS-PCR method for each of these alterations, suitable for the massive screening we have envisioned for AA and MDS.In BMF, we have first identified 21 MDS patients with known LGL and screened them for the presence of STAT3 mutations: in the CTLs of 6/21 of these patients STAT3 mutations were found and thus less clinically apparent LGL expansion could also be present in more classical MDS. Thus, we extended our screening to CTLs from an additional 368 patients with MDS and no suspected concomitant LGL: we identified 9 additional patients with STAT3 mutated clones. MDS patients carrying STAT3 mutant CTL clones had both advanced and low risk disease (low, n=1; int-1, n=4; int-2, n=8; High, n=2). These patients were characterized by a higher frequency of hypocellular bone marrow (55 vs. 10.5%; p<.001), and neutropenia at diagnosis (p<.04). No significant differences were found in overall survival. By analogy we also searched for STAT3 CTL clones in AA (N=148) and PNH (N=30). In total, we have identified 17 (10%) AA patients with STAT3 mutant CTL clones: all of these patients did not display manifest LGL. Clinically, these patients had a higher proportion of non severe AA (40% vs. 23%) and were more likely to respond to first line immunosuppression (76 vs., 65%) though no statistical significance was reached. In addition, cases with BMF and a subclinical mutant CTL clones were retrospectively tested for the presence of a TCR rearrangement: an oligoclonal (40% of cases) or monoclonal pattern (20% of cases) was seen in most of patients. In sum, the surprising discovery of STAT3 mutated clones in BMF states seems to be predominantly in AA patients and can be also found MDS cases with mainly, hypoplastic features, suggesting that STAT3 mutated self reactive CTL clones may play a role in immune pathogenesis of these conditions. Disclosures:Koskela:Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Mufti:Celgene: Consultancy, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Epigenetic Modulation of CD30 Gene Expression in Alemtuzumab Resistant T Cell Malignancy:Rationale and Efficacy of Brentuximab Vedotin.

Abstract 2454▪▪This icon denotes a clinically relevant abstract Background:T-Cell prolymphocytic leukemia (T-PLL) is an aggressive hematological disease mainly affecting adults. It is characterized by skin involvement and high lymphocyte counts. It affects mature T-cells mainly of the CD4+/CD8- phenotype. Although treatment with alemtuzumab, an anti-CD52 monoclonal antibody (mAb), induces complete remission (CR) in >50% of patients, it is short lived (median: 6 mos) and resistance is common. Epigenetic therapy with the histone deacetylase inhibitors (HDACi) vorinostat (SAHA) or romidepsin and the hypomethylating agent cladribine (2-CdA), can circumvent lack of response to alemtuzumab. Brentuximab Vedotin (SGN-35) is an antibody drug conjugate ADC) targeted against CD30 (TNFRSF8) and we have show here this ADC has activity in combination with epigenetic agents in T-PLL. Methods:Five T-PLL patients were studied. Diagnosis was made by hematopathology, flow cytometry and TCR rearrangement studies. Alemtuzumab (30 mg SQ d1,3,5) and 2-CdA (5mg/m2 IVd1–5), followed by alemtuzumab, 2-CdA and SAHA (400mg po daily X 5 days) or romidepsin (14mg/m2IV d 1,8,15) was given. After, epigenetic therapy with 2-CdA, SAHA, and romidepsin expression of CD30 in skin and blood was assayed where applicable.SGN-35 was administered at 1.8 mg/kg IV q 3 weeks. Peripheral blood was obtained and assayed under an IRB approved protocol. Genes of interest were analyzed by qRT-PCR. Chromatin Immunoprecipitation (ChIP) assays were used to study epigenetic changes at the CD30 promoter. Results:Of the 5 patients studied, three were alemtuzumab refractory with disease in blood and/or skin. With the addition of 2-CdA and SAHA or romidepsin, all three patients’ peripheral blood counts normalized and flow cytometry and PCR assays were negative for residual disease. One patient at the time of relapse developed skin lesions that were unresponsive to treatment with alemtuzumab, romedpsin, 2-CdA, or palatrexate. Lack of response to alemtuzumab in this patient was circumvented through induction of the cell surface receptor CD30, the target for the potent ADC, brentuximab vedotin. qRT-PCR assays on leukemic blood demonstrated that 4/5 of the T-PLL patients demonstrated increased CD30 mRNA following epigenetic therapy (mean: 14; range: 1.3–69 fold). These increases in CD30 mRNA expression correlated with increases in CD30 protein levels by Western blot analysis. CD30 and CD52 expression in the skin was confirmed by immunohistochemical analysis. In two patients, ChIP analysis of the CD30 promoter showed decreases in histone methylation markers and increases in Pol II binding at the coding sequence after treatment with epigenetic agents, consistent with increased CD30 expression. The patient with skin lesions had a dramatic response of his skin and normalization of his WBC count, with flow and PCR studies of skin and blood negative for residual disease after two cycles of SGN35.In contrast, a second patient with T-PLL who was refractory to up front alemtuzumab (IV) responded and achieved remission with the addition of 2-CdA, Her leukemic cells 2 days post treatment expressed CD30 mRNA in. At relapse 16 months later, her leukemic cells did not express CD30 and treatment with 2-CdA and romedepsin was unable to derepress CD30 gene expression. Correlative studies with ChiP assays differed from the previous patient. Further elaboration and follow up will be presented with both patients. Conclusions:Therapy with HDACi and 2-CdA epigenetically augments clinical activity of alemtuzumab and SGN-35, as evidenced by de-repression of TNFSFR8 (CD30) and increased response to treatment in one patient. While CD30 is a gene that can be epigenetically regulated in T cell malignancies, further complexities in epigenetic regulation of CD30 require additional investigation. Correlative studies are underway to elucidate the regulation of CD30 by epigenetic mechanisms. We present a rationale for the use of epigenetic drugs in combination with mAbs such as alemtuzumab and ADCs in T-PLL. A prospective multicenter clinical trial is warranted. Disclosures:Off Label Use: Brentuximab vedotin for PLL. Epner:Seattle Genetics: Speakers Bureau.

Primary CLL Xenograft: T-Cells Friend or Foe? the Role of T-Cells in the Primary CLL Xenograft Model.

AbstractAbstract 2888Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by the gradual accumulation of mature B cells in peripheral lymphoid organs. However, limited access to this proliferating tumour population in CLL patients and difficulties in modelling this proliferation in vitro necessitates the establishment of human xenograft models that can recapitulate the human disease. Several CLL xenograft models have already been established, however due to their inability to fully recapitulate human tissue distribution and their restricted period of engraftment, these models are not suitable for testing novel chemotherapeutics.To address this, modifications were made to a recent xenograft model established by Bagnara et al, utilising immuno-compromised NOG mice on a human haematopoietic microenvironment provided by prior propagation of human CD34+ umbilical cord blood cells. This was reported to reliably replicate the human disease but was of limited duration purportedly due to T-cell outgrowth.Our aim therefore was to determine the role various T-cells subsets play in engraftment and its demise in order to enhance the duration of the CLL engraftment in this primary CLL xenograft model. This was accomplished, by a) sequential cull of animals engrafted by primary CLL cells to monitor kinetics of T-cell engraftment over time, b) by addressing clonality and the potential anti-CLL effect of expanded T-cells and c) by depletion of implicated autologous T-cell subsets prior to their co-administration into mice with CLL tumour cells.The engraftment kinetics of patient PBMC in murine spleen; encompassing both T-cells and tumour CLL; were tracked and we observed that the relative proportions of patient T-cell subsets altered within 8 weeks following injection. Initially, during the first 6 weeks, there was rapid expansion of helper (CD4+) and regulatory (CD4+/CD25+) T-cells. Subsequently, their proportion decreased as the cytotoxic (CD8+) T-cells expanded becoming the dominant T-cell subtype. Interestingly, by using PCR amplification of TCR rearrangements we noted that these T-cells were not clonally expanded and that their co-incubation with autologous CLL cells in vitro failed to exert a cytotoxic effect.We have previously demonstrated that CLL engraftment could occur even if patient T-cells were depleted to a minimal level of 0.6–5×104 cells or 2% of initial T-cell numbers. Here, we demonstrate that depletion of either helper (CD4+) or regulatory (CD25+) T-cells resulted in a reduced level of engraftment in murine peripheral blood and spleen, extended graft duration and prolonged survival of the animals. In contrast, depletion of either cytotoxic (CD8+) or natural killer (CD56+) T-cells had limited effect on both engraftment initiation and duration of the graft (none<CD8<CD56<CD4<CD25<CD3, 101 vs 96 vs 115 vs 127 vs 130 vs 151 days).Taken together our results indicate a crucial role for helper (CD4+) and regulatory (CD25+) T-cell subsets in both initiation and termination of CLL engraftment. The expansion of CD8+ T-cells at the end of engraftment may be co-incidental and not a reflection of an anti-tumour effect. Further studies are necessary to address the exact interaction between patient CLL cells and regulatory T-cells. Finally, manipulation of the regulatory T-cell subset in this xenograft model can provide robust CLL proliferation amenable for drug testing. Disclosures:No relevant conflicts of interest to declare.

Thymic function is maintained during Salmonella-induced atrophy and recovery.

Thymic atrophy is a frequent consequence of infection with bacteria, viruses, and parasites and is considered a common virulence trait between pathogens. Multiple reasons have been proposed to explain this atrophy, including premature egress of immature thymocytes, increased apoptosis, or thymic shutdown to prevent tolerance to the pathogen from developing. The severe loss in thymic cell number can reflect an equally dramatic reduction in thymic output, potentially reducing peripheral T cell numbers. In this study, we examine the relationship between systemic Salmonella infection and thymic function. During infection, naive T cell numbers in peripheral lymphoid organs increase. Nevertheless, this occurs despite a pronounced thymic atrophy caused by viable bacteria, with a peak 50-fold reduction in thymocyte numbers. Thymic atrophy is not dependent upon homeostatic feedback from peripheral T cells or on regulation of endogenous glucocorticoids, as demonstrated by infection of genetically altered mice. Once bacterial numbers fall, thymocyte numbers recover, and this is associated with increases in the proportion and proliferation of early thymic progenitors. During atrophy, thymic T cell maturation is maintained, and single-joint TCR rearrangement excision circle analysis reveals there is only a modest fall in recent CD4(+) thymic emigrants in secondary lymphoid tissues. Thus, thymic atrophy does not necessarily result in a matching dysfunctional T cell output, and thymic homeostasis can constantly adjust to systemic infection to ensure that naive T cell output is maintained.

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

Author SummaryCertain lineages of T cells can recognize lipids as stimulatory antigens when presented in the context of CD1 molecules. We know how most Natural Killer T (NKT) cells react with this unusual ligand because they use a single invariant T cell receptor (TCR) alpha chain to do the job. NKT cells place particular emphasis on their CDR3α and CDR2β loops in recognition of antigen—these complementarity determining regions (CDRs) are the hypervariable parts of the TCR that “complement” an antigen's shape. How do these other T cells recognize closely related yet distinct lipid antigens? Here we show that human CD1d-restricted T cells, typically called Vα24− T cells due to their use of diverse Vα domains in their TCRs, use similar molecular strategies to respond to lipid antigens presented by CD1d. To this end we present a 2.5 Å complex structure of a Vα24− TCR complexed with CD1d presenting the protypical lipid, α-galactosylceramide (αGalCer). The TCR examined in this study notably shifts its binding slightly, placing more emphasis on the interaction with the CDR1α loop as revealed through alanine scanning mutagenesis. This shift explains the inability of these T cells to respond to lipids that vary at this site of contact (the 4'OH), like the related α-linked glucosylceramide. These results provide a molecular basis for the fine-specificity of different CD1d-restricted T cell lineages.

Expression and Function of PML-RARA in the Hematopoietic Progenitor Cells of Ctsg-PML-RARA Mice

Because PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice (KLS = Kit+Lin−Sca+, which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

Immune Recovery in Adult Patients after Myeloablative Dual Umbilical Cord Blood, Matched Sibling, and Matched Unrelated Donor Hematopoietic Cell Transplantation

Immunologic reconstitution after allogeneic hematopoietic cell transplantation is a critical component of successful outcome. Umbilical cord blood (UCB) transplantation in adult recipients is associated with slow and often inadequate immune recovery. We characterized the kinetics and extent of immune recovery in 95 adult recipients after a dual UCB (n=29) and matched sibling donor (n=33) or matched unrelated donor (n=33) transplantation. All patients were treated with myeloablative conditioning. There were no differences in the immune recovery profile of matched sibling donor and matched unrelated donor recipients. Significantly lower levels of CD3+, CD4+, and CD8+ T cells were observed in UCB recipients until 6 months after transplantation. Lower levels of regulatory T cells persisted until 1 year after transplantation. Thymopoiesis as measured by TCR rearrangement excision circle was comparable among all recipients by 6 months after transplantation. In a subset of patients 1 year after transplantation with similar levels of circulating T cells and TCR rearrangement excision circle, there was no difference in TCR diversity. Compared to HLA-identical matched sibling donor and matched unrelated donor adult hematopoietic cell transplantation recipients, quantitative lymphoid recovery in UCB transplantation recipients is slower in the first 3 months, but these differences disappeared by 6 to 12 months after transplantation.

Tcf-1 protects thymocytes from malignant transformation. (111.43)

Abstract The DNA binding protein Tcf-1 is a gatekeeper of lymphocyte fate, yet its molecular functions remain largely elusive. Tcf factors are mediators of Wnt/ β-catenin signaling, a pathway that is linked with many genomically unstable cancers. Here we provide evidence that Tcf-1 may play a crucial role in protecting the genome from damage and genomic instability. This is particularly important during TCR rearrangement when developing T-cells introduce damage to their DNA as a consequence of Rag activity. Interestingly, the Tcf-1 binding pattern on DNA as determined by ChIP-seq is highly correlated with that of Rag2. We further show that Tcf-1 binds several components of the DNA repair and remodeling machineries. Importantly, β-catenin outcompetes some of these interactions, with fatal consequences, as overt activation of β-catenin in murine thymocytes (CD4Cre;Ctnnb1ex3) causes Rag-dependent lymphomas that carry recurring TCRα ;Myc translocations. CD4Cre; Ctnnb1ex3 thymocytes also display increased chromatin accessibility (H3K4me3 and H3K9/14Ac), a risk-factor for translocations. In summary, our data point to novel roles for components of the Wnt/ β-catenin signaling pathway in defining chromatin structure, recombination and repair in general, and for the generation of recurring t(14;15)(C2;D1) translocations in particular, with implications for human malignancies in which analogous translocations have been described.

High-throughput sequencing of Memory and Naïve T-cell receptor repertoires at the RNA and DNA levels reveals differences in relative expression of expanded TCR clones (178.12)

Abstract Recent advances in high throughput sequencing technologies have enabled the profiling of TCR repertoires at an unprecedented level of resolution. Adaptive Biotechnologies has developed sequencing methods that are able to sequence TCR rearrangements by sequencing either using cDNA isolated from RNA, or using genomic DNA directly. We used Adaptive’s immunoSEQ technology to profile CD8+ Memory, CD8+ Naïve, and unsorted PBMC repertoires from 2 individuals using cDNA and genomic DNA isolated from the same starting population of cells. Using this method, it is possible to identify instances of either differential over- or under-expression of particular rearrangements, and to study the overall impact of starting material on sequencing results. Understanding the implications of assaying TCR repertoires at the genomic versus RNA level is important for the interpretation of experimental results, as well as for informing the design of immune profiling experiments that use high-throughput sequencing methodologies.

Clinical Impact of NOTCH1 and/or FBXW7 Mutations, FLASH Deletion, and TCR Status in Pediatric T-Cell Lymphoblastic Lymphoma

Pediatric T-cell lymphoblastic lymphomas (T-LBL) are commonly treated on T-cell acute lymphoblastic leukemia (T-ALL) -derived protocols. Therapeutic stratification based on response to the prephase treatment and on minimal residual disease assessment is well established in T-ALL but is not easy to extrapolate to T-LBL. The identification of molecular prognostic markers at diagnosis in T-LBL could provide an alternative for early therapeutic stratification. Our study determines the frequency and prognostic value of NOTCH1/FBXW7 mutations (N/F(mut)), FLASH deletion at chromosome 6q, and TCR rearrangements in a prospective cohort of pediatric T-LBL. Pathologic samples were obtained at diagnosis for 54 patients treated according to the EuroLB02 protocol in France. N/F(mut) were identified by direct sequencing and allelic dosage was used to detect FLASH and TCRγ deletions, which were interpreted in conjunction with TCRγ, TCRβ, and TCRδ rearrangements. N/F(mut) were found in 55% of T-LBL patients, in whom they were associated with improved event-free survival (P < .01) and overall survival (P < .01). FLASH monoallelic deletions were observed in 18% of patients; they were predominantly N/F wild-type (six of nine) and tended to be of inferior prognosis (P = .09). Absence of biallelic TCRγ deletion (ABD) was seen in 7%, all of which were N/F(mut) and identified a poor prognosis group (P = .02). On multivariate analysis of N/F(mut), TCRγ ABD, and FLASH deletion, only N/F(mut) was an independent factor for good prognosis. Mutational status of NOTCH1/FBXW7 represents a promising marker for early therapeutic stratification in pediatric T-LBL.

T-cell immunodeficiency and reconstruction based on TCR rearrangement analysis in hematological malignancy: update from 2011 ASH annual meeting

Poor cellular immune function may relate to carcinogenic processes and to worse prognosis in solid tumor patients as well as in leukemia. Moreover, the progression of tumor might further induce the cellular immune suppression. Therefore, a set of molecular immunological techniques to analyze and monitor the changes of host T-cell immune status is needed, which can fully characterize the feature of T-cell immunodeficiency in different malignancies, providing information and direction for immune reconstruction, in particular for enhancement the specific anti-tumor immune function.

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34⁺CD38dimLin⁻ cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34⁺CD38brightLin⁻ cells; (c) CD34⁺CD1a⁺CD11c⁻ cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34⁻CD1a⁺CD3⁻CD11c⁻ cells, which resemble CD4⁺CD8⁺ double-positive T cells in the thymus; and (e) CD34⁻CD1a⁺CD3⁺CD11c⁻ cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⁺ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre-T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.

Imatinib mesylate for lymphocytic variant of hypereosinophilic syndrome

To the editor: With a great interest I read a paper by Christoforidou et al. [1] showing the high efficacy of imatinib mesylate (IM) in a patient with L-HES and undetectable FIP1L1-PDGFRA (F/P) fusion transcript. However, after reading this case description, it seems that several issues need to be clarified and commented. The diagnosis of L-HES requires the detection of the abnormal T-cell phenotype in peripheral blood by flow cytometry and the presence of T-cell receptor clonal rearrangement [2]. It was demonstrated that clonal TCR rearrangement was detectable in patients with F/P mutation and treatment with IM resulted in disappearance of the clonal TCR pattern [3]. In this publication, the authors described a patient with lymphocytosis involving clonal but phenotypically normal CD41 cell population, but these cells were detected in marrow. I wonder if these cells were also found in peripheral blood? The presence of CD41 cells in patients with hypereosinophilic syndromes has already been demonstrated by other study groups, but it should be highlighted that this finding is extremely rare. The occurrence of such a T-cell population is usually associated with other features of T-cell mediated hypereosinophilia, e.g., increased serum IgE levels or cytokine and chemokine overproduction [4,5]. These results are needed to better characterize this variant but they are lacking in this report. Corticosteroids (CS) remain a treatment of choice for patients with L-HES [2]. The authors reported severe side effects after therapy with steroids so they were discontinued. It was demonstrated in most studies that CS resulted in decrease of blood eosinophilia and resolution of clinical symptoms but the proportion of abnormal T-cell remained unaffected [6,7]. In relation to this observation, I would expect such data from the authors of this presentation. We should keep in mind that treatment with IM, especially at a dose of 400 mg daily is expensive and associated with numerous side effects. Therefore, it seems rational to initiate IM only for patients who developed severe steroid-related complications and still require the continuation of treatment. Based on the presented case, it is difficult to judge that IM led to remission in a patient with L-HES. The therapy resulted in decrease of blood eosinophilia and withdrawal of some clinical symptoms, but in fact IM did not affect eosinophilic infiltration of the colon. The proportion of a clonal T-cell population remained intact. This case has shown that IM may remain an interesting therapeutic option but only for those patients who are resistant or intolerant to CS. As the abnormal T-cell subset remained unchanged after IM, we may expect that IM and CS will not prevent those patients from lymphoma transformation.

Linfoma intestinal de células T asociado a enteropatía y sin relación con enfermedad celíaca

Type II enteropathy-associated T-cell lymphoma (EATL) is an uncommon intestinal lymphoma. We report the case of a 73-year-old man with diarrhea and weight loss. Duodenal biopsy showed atrophy and infiltration of irregular lymphocytes. Immunohistochemistry was positive for CD3, CD8, and CD56 with monoclonal TCR rearrangement. The HLA-DQ genotype was DQ5/DQ9. The Epstein-Barr virus RNA test was negative. Before specific chemotherapy could be administered, the patient was admitted to hospital for a respiratory infection and died from a cause unrelated to his lymphoma. The differential diagnosis of CD56-positive lymphoproliferative processes include type II EATL, primary T-cell/natural killer-cell intestinal lymphoma and hepatosplenic T-cell lymphoma. The patient had CD8 y CD56+ markers that allowed type I EATL to be excluded. The HLA-DQ genotype did not correspond to celiac disease and the biopsy showed proliferation of lymphocytes with atypia. The primary intestinal T-cell/natural killer-cell lymphoma was characterized mainly by the absence of CD8 and monoclonal reassortment of the TCR present in this case.

Gaucher Disease and LGL Proliferations: Provocative Commonality?

Abstract 1100Large granular lymphocytes(LGL)which may be of T or NK phenotype, normally account for 10–15% of peripheral blood(PB)lymphocytes. T-LGL leukemia is a clonal expansion of antigen-primed competent cytotoxic-T-lymphocytes(CTL)which is unique in that these cells retain many phenotypic and functional properties of normal cytotoxic effector T cells. Sphingolipid-mediated signaling has been shown to have a role in long-term survival of CTLs.The diagnosis of T-LGL leukemia requires proof of clonality, yet clonal T-LGL proliferations may occur in normal individuals with autoimmune diseases, viral infections, B cell lymphoproliferations and post bone marrow transplantation rendering the differentiation of malignant (leukemic) from benign LGL expansions at times difficult. It has been suggested that T-LGL proliferations with polyclonal or oligoclonal TCR gene rearrangement may be a response to a stimulus before the emergence of a dominant clone.Gaucher disease (GD)is a lysosomal storage disease caused by mutations in the gene encoding acid β-glucocerebrosidase, leading to the accumulation of glucocerebroside in tissue macrophages (Gaucher cells) and resulting in hepatosplenomegaly, cytopenias and skeletal involvement. GD and T-LGL leukemia share strikingly similar clinical features, including cytopenias, splenomegaly, B cell dyscrasias, arthralgias, pulmonary hypertension (PHT), and increased association with neoplasms and autoimmune disorders. The etiology of PHT, B cell dyscrasias, and the increased frequency of neoplasms in GD remains unclear.Table 1 describes the characteristics of 4 GD patients suffering from severe cytopenias relative to the overall phenotype of their GD in whom LGL were found to comprise the majority of the PB lymphocytes. Patient #1: 31year-old female suffering from severe GD with massive splenomegaly and severe cytopenias but refuses Gaucher-specific therapy; Patient #2: 31year-old female has suffered from severe Gaucher disease since childhood, was splenectomized, has unexplained anemia and known LGL expansion for 7 years but clonality studies continue to be negative; Patient #3: 45 year-old female with severe GD, splenectomized, suffered from AVN, bone crisis autoimmune hemolytic anemia, PHT, and B cell dyscrasia; and Patient #4: 72 year-old male has asymptomatic GD with progressive pancytopenia who was diagnosed with myelodysplastic syndrome(MDS),T-LGL proliferation and monclonal gammopathy of unknown origin(MGUS). LGL cells comprised 70% of PB lymphocytes in addition there was severe trilineage dysplastic changes, TCR rearrangement was positive. Interestingly, 8 months after the start of enzyme replacement therapy(ERT),counts improved and the number of PB LGL was reduced to <10% of lymphocytes. In a cohort of 55 consecutive GD patients, 38% had LGL expansions in the PB smears. Since both conditions may be asymptomatic for many years, their co-existence can easily be missed.Table 1:Patient characteristics1234Age at diagnosis31314572GenderFemaleFemaleFemaleMaleSplenectomyNoYesYesNoBony diseaseAVNBone crisis, AVN, osteomyelitisNo?Pulmonary HypertensionNoNoYesNoAutoimmune phenomenonNoNoAIHANoB cell dyscrasiasYesNoPolyclonal hypergammagloblulinemiaMGUSOther neoplasmsNoNoNoMDSLeucocyte count (×10/ul)2.213.36.62.9LGL percent of lymphocytes52%62%30%70%LGL absolute count (×10/ul)0.55.111.10.532LGL phenotypeCD3+,CD8+, CD56+, CD16−, CD57−, TCR αβ+CD3+, CD8+, CD16−, CD56−, TCR αβ+?CD3+, CD8+, CD16−, CD56−, TCR αβ+2 populationsa) CD3+, CD8+, CD56+, TCR αβ+9.3g/dlb) CD3−,CD56+,CD4−, CD8−Hemoglobin (g/dl)4.911.311.4Platelet count (x10/ul)111779630TCR rearrangementNegativeNegativePositivePositiveBone marrow pattern of involvementMild interstitial infiltrate of small lymphocytes CD8+TMild interstitial infiltrate of small lymphocytes CD8+TERTNoYesYesYesERT: improvement of cytopeniasUntreatedNoNoYesThe co-existence of two hematological pathologies in more than one patient and the possibility that therapy for one may impact the other, is provocative. We suggest that LGL expansions in GD may be reactive to glucocereborside accumulation in Gaucher cells, and may play a pathogenic role in the development of some of the features of GD as well as induce/exacerbate some associated disorders. Disclosures:Zimran:Actelion: Honoraria.

Phenotypic and genetic characterization of adult T-cell acute lymphoblastic leukemia with del(9)(q34);SET-NUP214 rearrangement

SET-NUP214 rearrangement is a recently recognized recurrent chromosomal translocation mostly observed in T-ALL. In order to characterize this rare entity, we performed phenotypic and genetic characterization of SET-NUP214 rearrangement through an investigation of a series of 40 consecutive samples of adult T-ALL that was selected among 229 adult ALL cases during 4years in a single institution. Four cases (10%) of SET-NUP214 translocation were identified in our study. In all cases, diagnosis of T-ALL was established according to the World Health Organization (WHO) classification, and clonal TCR rearrangements were found. The immunophenotypic markers were indicative of the precursor nature of T lymphoblasts, and they expressed one or both of the myeloid-associated antigens (CD13, CD33). Conventional cytogenetic analysis revealed complex chromosomal aberrations in all four SET-NUP214 rearranged cases and del(12)(p13)/ETV6 was frequently involved. Array-CGH demonstrated additional genomic imbalances in addition to deletion 9q34. The genomic breakpoint sequencing identified breakpoints at SET intron 7 and NUP214 intron 17, and random nucleotide addition was found in two cases at the site of rearrangement. Our independently derived data set from a single institution confirms previous findings of SET-NUP214 rearrangement, indicates the relatively high incidence of SET-NUP214 rearrangement in adult T-ALLs, and also demonstrates comprehensive clinical, phenotypic, and genetic characteristics of this entity. Also, our report on genomic breakpoints demonstrates the homogeneity in the localization of the genomic breakpoints at 9q34. Concurrent chromosomal aberrations identified in this study should provide further areas of interest in investigation of SET-NUP214-mediated leukemogenesis.