T-lymphoblastic Leukemia/lymphoma Research Articles

Flow Cytometric Evaluation of Surface and Cytoplasmic TRBC1 Expression in the Differential Diagnosis of Immature T-Cell Proliferations.

Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions. Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated. All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality. Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations.

T-lymphoblastic leukemia/lymphoma with interfollicular growth pattern and Castleman-like morphologic features

Indolent T-lymphoblastic proliferation (iT-LBP) is a recently recognized benign pathologic condition commonly associated with Castleman disease. The lymph node of this condition shows preserved lymphoid architecture with proliferation of nonclonal T-lymphoblasts in the interfollicular area. iT-LBP is a rare condition and a diagnosis of exclusion. As a rule, it requires sophisticated techniques to exclude T-lymphoblastic leukemia/lymphoma (T-LBL/L). We present an unusual T-LBL/L in a lymph node mimicking iT-LBP. The case showed preserved nodal architecture, morphologic features of hyaline vascular Castleman disease, proliferation of T-lymphoblasts in the interfollicular area, and no clonal T-cell receptor gene rearrangement. The follow-up lymph node specimen excised 6 months later showed full-blown lymphoblastic lymphoma with an abnormal karyotype, near-early-T-precursor phenotype, partial CD19 expression, and SETD2 mutation.

Comprehensive Evaluation and Validation of a Next-Generation Sequencing Assay for Minimal Residual Disease Detection in T-Lymphoblastic Leukemia/Lymphoma

Introduction: Detection of minimal residual disease (MRD) in T-lymphoblastic leukemia/lymphoma (T-ALL) has been used in post-therapy monitoring and outcome prediction. Currently, multi-color flow cytometry is widely used for MRD detection with limit of detection (LOD) of 0.1 to 0.01% depending on the clinical implementation. However, flow cytometry assays require extensive training and experience for accurate interpretation and standardization is challenging. Next generation sequencing (NGS) assays with panels targeting the inherent polymorphism of the immunoglobulin genes have been applied for MRD detection in B cell neoplasms, including an assay for myeloma and B-ALL MRD detection developed by Adaptive Biotechnologies (Seattle, WA) that was granted FDA approval for clinical testing. We have published a similar approach for T-ALL MRD detection using the polymorphic T-cell receptor (TCR) genes. These assays offer higher sensitivity, easier standardization, and more objective interpretation. Here we present our comprehensive evaluation and validation of a T-ALL MRD assay using the Adaptive Biotechnologies immunoSEQ kit for potential clinical application. Methods: NGS was performed using the Adaptive Biotechnologies immunoSeq kit and illumina NextSeq 500 according to manufacturer's instruction. Quantification of T cell repertoire was performed using the Adaptive Biotechnologies analysis pipeline. A single TCRβ sequence with ≥19% frequency of the T-cell population in diagnostic samples was defined as a T-ALL clone. Positive MRD was defined as detection of one or more such T-ALL sequences having a 100% DNA sequence match in the corresponding MRD samples. T-ALL leukemic cells and normal T cells were sorted from 2 bone marrow aspirates and peripheral blood from 20 healthy donors, respectively. T-ALL cells were spiked into pooled normal T cells to create a series of diagnostic T-ALL standards and MRD T-ALL standards down to 0.001%. Prior to assaying the standards, a T-ALL specimen was assessed at 11 different DNA input levels ranging from 3-1000ng, and interference of non-T-cell DNA was tested. For all other assays, 800ng DNA was used in two independent hsTCRB replicates for T-ALL clone identification in diagnostic samples and 2,400ng DNA was used in six independent hsTCRB replicates for T-ALL clone detection in MRD samples. Within run precision was assessed by assaying all standards and 3 pairs of diagnostic and MRD samples in triplicate. Between run precision was assessed by assaying 1 standard and 4 samples on two different days by the same technologist. Intermediate precision was assessed by assaying 3 pairs of diagnostic and MRD samples by two different technologists. LOD was determined as the lowest frequency at which MRD could be detected in MRD standards and Limit of Quantitation (LOQ) determined as the level below which the coefficient of variation (CV) >25% in MRD standards. Results: The %CV for T-ALL quantification across the range of DNA input was 4.5%, suggesting independence of quantification from DNA input amount. Non-T-cell DNA has almost no impact on T-ALL quantification. T-ALL clones were detected with 100% concordance in diagnostic and MRD standards. The %CV of within and between run precision and intermediate precision were < 25% in T-ALL identification assay and all MRD detection assays except at 0.001% MRD, in which %CV was 43.8%. Quantification of T-ALL in both diagnostic and MRD standards were highly correlated with expected values (R2 = 0.96, slope = 0.89 and R2 = 1.00, slope = 0.88, respectively). T-ALL identification and MRD detection in specimens stored at 4°C or at room temperature for up to 14 days were within 25% CV as compared to fresh specimens. The assay was then performed on 40 diagnostic clinical T-ALL samples and T-ALL clonal sequences were identified in 34 samples (85%). Conclusions: The immunoSEQ assay is evaluated and technically validated for clinical application, but requires validation in a larger cohort of clinical samples with concurrent outcome data. The assay is suitable for roughly 70% T-ALL where TCRβ is rearranged. The assay LOD for T-ALL MRD detection is 0.001% and LOQ is 0.01%, offering the possibility of improved sensitivity and more objective interpretation. Disclosures No relevant conflicts of interest to declare.

T- Cell Lymphoblastic Lymphoma/Leukemia: A Case Report

T-lymphoblastic leukemia/lymphoma (T-LBL) is rare, aggressive neoplasm of lymphoblasts committed to T-cell lineage involving bone marrow (BM), blood or presenting as a tissue-based mass involving the thymus, lymph nodes or extra nodal sites.

Flow cytometric characterization of acute leukemia reveals a distinctive "blast gate" of murine T-lymphoblastic leukemia/lymphoma.

Immunophenotypic analysis using multiparameter flow cytometry is an indispensable tool for diagnosis and management of acute leukemia. Mouse models have been widely used for medical research for more than 100 years and are indispensable for leukemia research. However, immunophenotypic analysis of murine leukemia was not always performed in published studies, and blast gating for isolation of blasts was shown only in very few studies. No systemic characterization of all types of murine acute leukemia in large cohorts by flow cytometry has been reported. In this study, we used flow cytometry to comprehensively characterize murine acute leukemia in a large cohort of mice. We found that murine T-lymphoblastic leukemia/lymphoma (T-ALL) exhibits a distinctive “blast gate” (CD45bright) with CD45/side scatter gating that differs from the “blast gate” (CD45dim) of human T-ALL. By contrast, murine B-lymphoblastic leukemia and acute myeloid leukemia show the same blast region (CD45dim) as human leukemia. Using blast cell gating, we for first time detected T-ALL development in FLT3-ITD knock-in mice (incidence: 23%). These leukemic cells were selectively killed by the FLT3 inhibitors crenolanib and midostaurin in vitro. These data suggest that FLT3-ITD plays a potential role in the pathogenesis of T-ALL and that FLT3-ITD inhibition is a therapeutic option in the management of patients with T-ALL. Our gating strategy for immunophenotypic analysis can be used for leukemogenesis and preclinical gene therapy studies in mice and may improve the quality of such analyses.

Prognostic Value of Recurrent Molecular Genetics and Epigenetics Abnormity in T Lymphoblastic Lymphoma / Leukemia -Review

T lymphoblastic lymphoma / leukemia is a strong invasive and has a high incidence of various molecular genetic abnormalities. The NOTCH1 / FBXW7 mutation is one of the most common mutations, and related with good prognosis in T-LBL / ALL. PTEN mutation, a poor prognostic factor, could be overcome by NOTCH1 mutations in pediatric patients to some extent. Patients with MLL gene abnormality and loss of heterozygosity 6q have worse prognosis than those with normal karyotype. The incidence of MLL gene abnormality, RUNX1 mutation and DNMT3A mutation in early precursor T-lymphoblastic leukemia was higher than that of other mature subtypes, which could be used as risk stratification factors. Epigenetic abnormalities play an important role in the malignant transformation of stem cells. Epigenetics treatment such as demethylation therapy may improve the prognosis of high-risk patients. In this article, the NOTCH1,FBXW7,PTEN and RAS gene mutations and prognosis, the molecular and cellular abnormalities and prognosis, as well as epigenetic abnormality and prognosis are reviewed.

Upregulated Neuro-oncological Ventral Antigen 1 (NOVA1) Expression Is Specific to Mature and Immature T- and NK-Cell Lymphomas.

Background:Recent studies have revealed that the splicing factor neuro-oncological ventral antigen 1 (NOVA1) is enriched in fibroblasts and accumulated T cells of tertiary lymphoid structures. In the present study, we investigated NOVA1 expression in various subtypes of mature and immature T- and natural killer (NK)-cell lymphomas as well as in various B-cell lymphoma subtypes. Methods:NOVA1 immunoexpression was evaluated in hyperplastic palatine tonsils (n = 20), T- and NK-cell lymphomas (n = 177), diffuse large B-cell lymphomas (n = 151), and other types of B cell lymphomas (n = 31). Nuclear staining intensity and percentage of positive tumor cells were graded. NOVA1 mRNA expression was analyzed in various lymphoma cell lines.Results:Tumor cells of T- and NK-cell lymphomas showed higher expression levels of NOVA1 than did normal paracortical T cells, and 56.5% of T- and NK-cell lymphoma cases showed diffuse and strong expression. The NOVA1 expression level varied according to the subtype; it was higher in angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL), and T lymphoblastic leukemia/lymphoma (T-LBL), but it was lower in ALK-positive ALCL. In almost all B-cell lymphomas, NOVA1 expression was very low or negative. NOVA1 mRNA was also expressed in Jurkat, a T-LBL cell line.Conclusions:The present findings suggest that NOVA1 upregulation may be involved in certain subtypes of T- and NK-cell lymphomas, but not in B-cell lymphomas. Upregulated NOVA1 expression seems to be a specific biological feature of activated T cells such as T- and NK-cell lymphomas.

LMO2 Is a Specific Marker of T-Lymphoblastic Leukemia/Lymphoma.

The diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) involving the thymus can be difficult to establish since neoplastic T lymphoblasts show significant phenotypic overlap with both normal thymocytes and thymocytes from epithelial thymic neoplasms (thymomas). LIM Domain Only 2 (LMO2) gene translocations have been implicated in the pathogenesis of a small subset of T-ALLs, and LMO2 protein has recently been reported to be expressed in a large proportion of T-ALLs. In this study, we tested specificity of LMO2 for distinction between neoplastic and nonneoplastic T-precursor cells in thymus and bone marrow. Our findings show that LMO2 is expressed in neoplastic lymphoblasts of T-ALL and is absent in thymocytes of normal thymuses or thymomas. LMO2 is therefore a useful marker for immunophenotypic assessment of thymic neoplasms.

The Role of Interim F-18 FDG PET/CT in Patients of T Lymphoblastic Leukemia/Lymphoma Treated with ALL-Type Regimens

Background:T lymphoblastic leukemia/lymphoma (T-LBL) is highly aggressive. Although intensive chemotherapies such as acute lymphoblastic leukemia (ALL)-type regimens are commonly used, about half adult patients eventually relapse and die of T-LBL. Overwhelming evidences have confirmed the role of interim F-18 FDG PET/CT in Hodgkin lymphoma and many ongoing studies have implemented risk-adapted strategy determined by interim FDG-PET/CT. However, the role of interim FDG-PET/CT in T-LBL remains unclear.Materials and methods:47 adult patients of T-LBL treated with ALL-like regimens were retrospectively reviewed. They were treated with modified Berlin-Frankfurt-Münster (BFM)-90 regimen (n=27), Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 regimen (n=5), hyperCVAD/MA regimen (n=12), or other unspecified regimens (n=3). Interim (defined as the period between induction therapy and re-induction therapy) FDG-PET/CT was done in all 47 patients and evaluated according to the International Harmonization Project (IHP) criteria.Results:The male to female ratio was 3:1, and 29.8% of patients were older than 30 years old. About 90% of patients had mediastinal mass at presentation and 53.2% had bone marrow infiltration. 85.1% of patients had advanced disease. After induction therapy, interim FDG-PET/CT was positive in 19 patients (40.4%), most of whom had residual disease in the mediastinal lesion. Subsequent treatments were not changed according to this interim PET/CT results. After a median follow up time of 28 months, the 2-year and 3-year progression free survival (PFS) rate were 39% and 30%, respectively, and the 2-year and 3-year overall survival (OS) rate were 54% and 45%, respectively. Using Kaplan-Meier survival analysis and log-rank test, it was found that interim FDG-PET/CT positivity correlated with significantly inferior PFS and OS (p=0.002 and 0.010, respectively). Furthermore, patients with higher age (>30) had inferior PFS and OS than younger patients (p=0.037 and 0.036, respectively). However, there were no significant relationship between PFS, OS and bone marrow infiltration, LDH level, and stages (p>0.05).Conclusions:Interim FDG-PET/CT may predict PFS and OS in adult patients of T-LBL treated with ALL-type regimens, which needs to be validated in prospective clinical trials. The optimal criteria for interim FDG-PET/CT evaluation and risk-adapted treatment strategy determined by interim FDG-PET/CT should be investigated in future clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Peripheral T-Cell Lymphoma Arising in a Patient With Prior T-Cell Acute Lymphoblastic Lymphoma: A Dyadic Case Series

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of T-cell lymphomas that portend a variable prognosis. Nonhepatosplenic/noncutaneous (NHNC) γδ-PTCLs are rare. Described here is an unusual case of transformation from T-lymphoblastic leukemia/lymphoma (T-ALL) to NHNC γδ-PTCL, not previously reported, as well as a case of PTCL arising in a patient with T-ALL. Patient 1, age 31, was diagnosed with T-ALL and treated with …

Inhibition of type I insulin-like growth factor receptor tyrosine kinase by picropodophyllin induces apoptosis and cell cycle arrest in T lymphoblastic leukemia/lymphoma

It has been recently shown that the type I insulin-like growth factor receptor (IGF-IR) contributes significantly to the survival of T lymphoblastic leukemia/lymphoma (T-LBL) cells, and it was therefore suggested that IGF-IR could represent a legitimate therapeutic target in this aggressive disease. Picropodophyllin (PPP) is a potent, selective inhibitor of IGF-IR that is currently used with notable success in clinical trials that include patients with aggressive types of epithelial tumors. In the present study, we tested the effects of PPP on Jurkat and Molt-3 cells; two prototype T-LBL cell lines. Our results demonstrate that PPP efficiently induced apoptotic cell death and cell cycle arrest of these two cells. These effects were attributable to alterations of downstream target proteins. By using proteomic analysis, seven different proteins were found to be affected by PPP treatment of Jurkat cells. These proteins are involved in various aspects of cellular metabolism, cytoskeleton organization and signal transduction pathways. The results suggest that PPP affects multiple signaling molecules and inhibits fundamental pathways that control cell growth and survival. Our study also provides novel evidence that PPP could be potentially utilized for the treatment of aggressive T-LBL.

Unusual T-lymphoblastic blast phase of chronic myelogenous leukemia.

T-lymphoblastic leukemia/lymphoma (T-ALL) presenting as blast phase of chronic myelogenous leukemia (CML-BP) is rare. In patients without history of CML, it is difficult to differentiate between CML-BP or de novo T-ALL. Here we reported 2 unusual cases of T-ALL presenting as CML-BP. Case 1 was a 24-year-old female with leukocytosis. Besides T-lymphoblasts (32%), her marrow exhibited some morphologic features of CML. Multiple remission or relapsing marrow had never demonstrated morphologic features of CML. Despite of imatinib treatment and stem cell transplant, she died 2.5 years later. Case 2, a 66-year-old male with diffuse lymphadenopathy, showed T-ALL in a lymph node and concurrent CML chronic phase (CML-CP) in his marrow. Same BCR-ABL1 fusion transcript with minor breakpoint was present in both the lymph node and marrow specimens. Although both cases did not have a history of CML, both cases represented T-lymphoblastic CML-BP with unusual features: Case 1 is unusual in that it presented as T-ALL with some CML morphologic feature but never showed CML-CP in her subsequent marrows biopsies; Case 2 is the first reported case of T-lymphoblastic CML-BP harboring BCR-ABL1 transcript with a minor breakpoint.

T-cell lymphoblastic leukemia/lymphoma: relapse 16 years after first remission.

Little information is available regarding late relapse in patients with T-lymphoblastic leukemia/lymphoma (T-LBL). Because of the aggressive nature of this disease, relapse is common and often happens early. Late relapses are rare and generally occur within a few years after initial remission. The relapse rate after 3 years has been reported to steadily decrease over time yet does not parallel with cure. We report a case of a 26-year-old female with T-LBL and relapse 16 years after her first remission with successful treatment with HyperCVAD and L-asparaginase.

Whole Exome Sequencing In Relapsed Pediatric T-ALL: Progression Into Relapse Is Characterized By An Increased Number Of Somatic Mutations and a Conservation Of Mutations In Leukemogenic Driver Genes

Acute precursor T-lymphoblastic leukemia (T-ALL) remains a serious challenge in pediatric oncology, because relapses carry a particularly poor prognosis with high rates of induction failure and death despite generally excellent treatment responses of the initial disease. It is critical, therefore, to understand the molecular evolution of pediatric T-ALL and to elucidate the mechanisms leading to T-ALL relapse and to understand the differences in treatment response between the two phases of the disease. We have thus subjected DNA from bone marrow samples obtained at the time of initial diagnosis, remission and relapse of 14 patients to whole exome sequencing (WES). Eleven patients suffered from early relapse (duration of remission 6-19 months) and 3 patients from late relapse (duration of remission 29-46 months).The Agilent SureSelect Target Enrichment Kit was used to capture human exons for deep sequencing. The captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al., Nucl. Acids Res., 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, Genome Res. 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al., Bioinformatics, 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible somatic driver mutations, candidate SNVs were filtered for non-synonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 50, and requiring absence of segmental duplications. Leukemia-specific mutations were identified by filtering against the corresponding remission sample and validated by Sanger sequencing of the genomic DNA following PCR amplification. We identified on average 9.3 somatic single nucleotide variants (SNV) and 0.6 insertions and deletions (indels) per patient sample at the time of initial diagnosis and 21.7 SNVs and 0.3 indels in relapse. On average, 6.3 SNVs were detected both at the time of initial diagnosis and in relapse. These SNVs were thus defined as leukemia specific. Further to SNVs, we have also estimated the frequency of copy number variations (CNV) at low resolution. Apart from the deletions resulting from T-cell receptor rearrangement, we identified on average for each patient 0.7 copy number gains and 2.2 copy number losses at the time of initial diagnosis and 0.5 copy number gains and 2.4 copy number losses in relapse. We detected 24/27 copy number alterations both in initial diagnosis and in relapse. The most common CNV detected was the CDKN2A/B deletion on chromosome 9p. Nine genes were recurrently mutated in 2 or more patients thus indicating the functional leukemogenic potential of these SNVs in T-ALL. These recurrent mutations included known oncogenes (Notch1), tumor suppressor genes (FBXW7, PHF6, WT1) and genes conferring drug resistance (NT5C2). In several patients one gene (such as Notch 1, PHF6, WT1) carried different mutations either at the time of initial diagnosis and or in relapse, indicating that the major leukemic clone had been eradicated by primary treatment, but that a minor clone had persisted and expanded during relapse. The types of mutations did not differ significantly between mutations that were either already present at diagnosis or those that were newly acquired in relapse, indicating that the treatment did not cause specific genomic damage. We will further characterize the clonal evolution of T-ALL into relapse by targeted re-sequencing at high depth of genes with either relapse specific or initial-disease specific mutations. In conclusion, T-ALL relapse differs from primary disease by a higher number of leukemogenic SNVs without gross genomic instability resulting in large CNVs. Disclosures:No relevant conflicts of interest to declare.

Abstract 1179: Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience.

Abstract Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and young adulthood, with a 2:1 male predominance; it comprises 15 and 25 percent of childhood and adult ALL, respectively, and 2 percent of adult non-Hodgkin lymphoma. Nelarabine is an anticancer prodrug of arabinofuranosylguanine (ara-G); it inhibits DNA synthesis and leads to high molecular weight DNA fragmentation and cell death. Nelarabine has showed relevant efficacy in phase II clinical trials, both in pediatric and in adult LBL/ALLpopulations. Design and Methods. We report clinical outcome results of 9 newly diagnosed and younger than 60 years T-ALL patients (median age 29 years, range 22-45 years, 3/6 M/F, 8 T-ALL, 1 T-LBL) treated according to pediatric-like adapted schedule. Cytogentics data and molecular biologic features will be provided on site. Induction cycle included Vincristine, daunoblastine, L-asparaginase and Prednisone. After induction, all the patients received consolidation therapy with cyclophosphamyde, L-asparaginase, Cytarabine and 6-Mercaptopurine. Subsequently all the patients receveid Nelarabine 1500 mg/sqm (days 1-3-5 every 21) for two cycles. All the patients shared the same strategy for intensification, which consisted in allogenic stem cell transplantation, if available, or additional courses of consolidation chemotherapy. Durations of complete remission (CR) and overall survival (OS) were estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of disease recurrence. Results. After a single induction course, 9/9 patients obtained a CR (100%). Eight patients underwent an allogenic bone marrow transplantation. After a median follow-up of 24 months, 7/9 patients (78%) are alive in CR. The median CR duration and OS were 13.4 and 24.4 months, respectively. Neurological toxicity of grade 3 has not been reported. We did not observe grade 3-4 haematological toxicity. Conclusion. Nelarabine is a promising drug, which induces a remarkable complete remission rate at the expense of a very low and manageable toxicity. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, University of Bologna and BolognAIL. Citation Format: Chiara Sartor, Mariachiara Abbenante, Cristina Papayannidis, Ilaria Iacobucci, Stefania Paolini, Emanuela Ottaviani, Annalisa Lonetti, Sarah Parisi, Anna Ferrari, Viviana Guadagnuolo, Sandro Grilli, Nicoletta Testoni, Michele Cavo, Giovanni Martinelli. Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2013-1179

Genome-Wide Analysis of NOTCH1, ETS Family Factors, and RUNX1 Binding in Human T Lymphoblastic Leukemia Cells Reveals Distinct Regulatory Elements

Abstract 1277NOTCH1 regulates gene expression by forming transcription activation complexes with the DNA-binding factor RBPJ and gain-of-function NOTCH1 mutations are common in human and murine T lymphoblastic leukemia/lymphoma (T-LL). Via ChIP-seq studies of T-LL cells with constitutive Notch activation, we previously showed that NOTCH1/RBPJ binding sites in T-LL genomes are highly enriched for motifs corresponding to Ets factors and Runx factors. In this study, we determined the relationship of NOTCH1, RBPJ, ETS1, GABPA and RUNX1 binding sites in human T-LL cells by performing ChIP-Seq for each of these factors, as well as the chromatin marks H3K4me1, H3K4me3, and H3K27me3, and aligning the resulting sequences to human genome reference hg19 using programs available through Cistrome. Peak calling was performed with MACS2, and motif analysis was performed using SeqPos, which relies on JASPAR, TRANSFAC, Protein Binding Microarray (PBM), Yeast-1-hybrid (y1h), and human protein-DNA interaction (hPDI) databases to find known motifs and can also perform de novo motif discovery. Our analysis showed even more pervasive overlap of NOTCH1/RBPJ binding with ETS1/GABPA and RUNX1 factor binding than was predicted by motif analysis, in part due to binding of Ets factors and RUNX1 to non-canonical sequences. Heat-map analysis with K-means clustering on NOTCH1 binding regions identified three major classes of RBPJ/NOTCH1: class 1, characterized by high NOTCH/RBPJ signals, binding of the cofactors ZNF143, ETS1 and GABPA, high H3K4me3 signals, localization to promoters, and binding motifs for ZNF143; class 2, characterized by low NOTCH/RBPJ signals, binding of the cofactors ETS1, GABPA and RUNX1, high H3K4me3 signals, and Ets factor and CREB binding motifs; and class 3, characterized by high NOTCH/RBPJ signals, binding of RUNX1 and ETS1 cofactors, high H3K4me1 signals, intergenic localization (consistent with enhancers), and motifs for RUNX factors, ETS factors, and RBPJ. Of note, the nearest binding sites to the most responsive NOTCH1 target genes (defined as >2 fold stimulation when NOTCH1 was activated following release of gamma-secretase inhibitor (GSI) blockade by drug washout) were preferentially associated with Class 3 sites. Furthermore, shRNA knockdown of Ets factors and RUNX1 in T-LL cell lines induced apoptosis and reduced cell proliferation, implicating these factors in maintenance of T-LL growth and survival. Combination of knockdown of either Ets factors or RUNX1 with GSI treatment resulted in more severe phenotype in terms of apoptosis and cell growth compared to the knockdown or GSI treatment alone. In summary, our studies represent a step forward towards genome-wide understanding of how Notch works in concerts with other transcription factors to regulate the transcriptome of T-LL cells. Disclosures:No relevant conflicts of interest to declare.

Delayed-type Hypersensitivity in Response to L-asparaginase in a Case of Acute Lymphoblastic Leukemia

L-asparaginase (L-Asp) is an important reagent for acute lymphoblastic leukemia because asparagine is required for the malignant growth of tumor cells, especially lymphoblastic leukemia cells. An allergic response to L-Asp is not unusual because L-Asp is derived from Escherichia coli and is often recognized as a foreign protein. The hypersensitivity induced by L-Asp is of the immediate type in most cases. We report on a 5-year-old girl who was hospitalized for precursor T-cell lymphoblastic leukemia. She was treated according to a Tokyo Children's Cancer Study Group protocol (TCCSG ALL L09-1603 HEX/BFM). During the intensification phase, blisters with erythema developed on the arm proximal to the catheter insertion site owing to a delayed-type hypersensitivity reaction caused by intravenous L-Asp administration. She was treated with additional methylprednisolone, tapered dexamethasone, and an antihistamine for the allergic reaction. No asparaginases other than E. coli L-Asp have been approved for use in Japan. Other asparaginases, such as polyethylene glycol L-Asp and Erwinia L-Asp should be quickly approved for use as alternative chemotherapy reagents in Japan.

Distinct immunophenotype of early T-cell progenitors in T lymphoblastic leukemia/lymphoma may predict FMS-like tyrosine kinase 3 mutations

FMS-like tyrosine kinase 3 (FLT3) mutation in T lymphoblastic leukemia/lymphoma (T-LL) is rare (∼4%) and reported only in cases with CD117 expression. This study aimed to identify the immunophenotypic features that may predict FLT3 mutations. We report 3 (43%) of 7 CD117(+) T-LL cases harboring FLT3-internal tandem duplication mutation. Compared with 4 FLT3-unmutated cases, all 3 FLT3-mutated cases had a distinct immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(uniform+)/Tdt(+)) corresponding to the stage of earliest thymic T-cell progenitors possessing myeloid lineage potential. Indeed, all FLT3-mutated T-LL cases expressed myeloperoxidase on a very small subset of blasts and, thus, may be further considered a mixed phenotype acute leukemia, T/myeloid, by the 2008 World Health Organization classification scheme. We conclude that this unique immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(+)/Tdt(+)) is a better predictor of FLT3 mutation than sole CD117 expression.

Composite T lymphoblastic leukemia/lymphoma and diffuse large B-cell lymphoma: Case report

This report concerns a unique case of a composite lymphoma composed of T-lymphoblastic leukemia/lymphoma (T-LBL) and diffuse large B-cell lymphoma (DLBCL) in a 72-year-old woman with generalized lymphadenopathy, splenomegaly and ascites. Laboratory findings showed increased lactate dehydrogenase and soluble interleukin-2 receptor. The biopsy specimen showed replacement of the normal architecture of the lymph nodes by a tumor containing a dual cell population composed of large lymphocytes and medium-sized lymphocytes. Sheets of large lymphocytes often were punctuated by clusters of medium-sized lymphocytes. Flow cytometry and immunohistochemical analysis showed a composite lymphoma with both T-LBL and DLBCL. The T-LBL expressed CD1a, CD3, CD4, CD8, and terminal deoxynucleotidyl transferase. The DLBCL expressed CD19 and CD20, CD23, bcl-2, bcl-6, MUM1 and immunoglobulin κ light chain. Polymerase chain reaction detected a monoclonal pattern of T-cell receptor γ and immunoglobulin heavy chain rearrangements in the same specimen. She received eight cycles of R-CHOP (rituximab+cyclophosphamide, doxorubicin, vincristine, prednisone) therapy and achieved complete remission. She has shown no signs of recurrence 20 months after the diagnosis. We describe here a very unusual and, to the best of our knowledge, an as yet never reported case of a primary composite lymphoma of T-LBL and DLBCL.

Inactivation of INK4a/ARF Accelerates the Development of PTEN Deficient T-ALL

AbstractAbstract 4197 Introduction:Acute T-lymphoblastic leukemia/lymphoma (T-ALL) is a group of heterogeneous disease with deregulated expression of variable types of genes. PTEN and INK4a/ARF are among the most frequently inactivated tumor suppressors in human T-ALL. The PTEN gene encodes a phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway activity. PI3K activates a variety of key signaling proteins that are critical in cell cycle control and cell survival. The Ink4a/Arf gene encodes two distinct tumor suppressors, p16 INK4a and p14 ARF (p19ARF in mice), that function as regulators of the pRB and p53 pathways, respectively. Since dual inactivation of PTEN and INK4a/ARF tumor suppressor genes occurs relatively frequent in human T-ALL, we investigated the specific role and inter-relationship of PTEN and INK4a/ARF tumor suppressor genes in the pathogenesis of T-ALL in genetically engineered mouse models (GEMMs). Methods:We have intercrossed the Ptenflox/flox mice with Ink4a/Arfflox/flox mice, generating compound conditional KO mice carrying both Ptenflox/flox and Ink4a/Arfflox/flox alleles. The compound Ptenflox/flox and Ink4a/Arfflox/flox mice were further intercrossed with Lck-Cre mice, an extensively characterized transgenic mouse strain that expresses Cre recombinase under the T-cell specific LCK promoter. The Lck-Cre transgenic mice express Cre in T-lymphoid cells, including T-lymphoid cells at early developmental stage. Serial analyses from birth to 8 months of age (9 time points) and/or end-point study were performed for a phenotypic workup. Kaplan-Meier curves for overall and tumor-free survival were generated at the end of the study. Grossly normal or tumor-bearing tissue was harvested for histopathlogical and molecular analyses. Results:Molecular analyses showed expected recombinant alleles and absence of Pten and INK4a/ARF proteins in mouse embryonic fibroblasts (MEFs) and T-cells after Cre-mediated excision with concurrent increased expression of phosphorylated Akt indicating the proper deletion of the Pten tumor suppressor gene. Mice with T-cell specific deletion of either Pten, Ink4a/Arf or Pten and Ink4a/Arf were closely monitored closely for perturbations in the hematolymphoid system. The mean disease-free survival (lack of palpable adenopathy) of tInk4a/Arf-/- and tPten-/-, littermates averaged 53, 30 weeks, respectively, while compound and tInk4a/Arf-/- and tPten-/- mice succumbed between 6 and 24 weeks (mean 12 weeks; p<0.001) to a rapidly progressive disease characterized by generalized wasting, mediastinal mass and enlarged lymph nodes. Histologically, the neoplastic cells are immature lymphoid cells, involving multiple organs and circulating blood. Immunophenotypic studies by flow cytometry demonstrated immature T-cell lineage cells (T-ALL).Conclusions: Using our genetically engineered mouse models, we have provided genetic evidence that INK4a/ARF and PTEN cooperate in constraining the development of T-ALL. These mouse models provide a platform for discovering new molecular targets for T-ALL therapy preclinically. Ongoing efforts focus on further charactering the difference in pathology, the PI3K/AKT pathways, DNA damage, cell cycle control, and drug sensitivities among the various T-ALL models. Disclosures:No relevant conflicts of interest to declare.