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Refractory/relapsed (R/R) diffuse large B-cell lymphoma (DLBCL) patients' failure of salvage chemotherapy had extremely worse prognoses. Herein, 14 R/R DLBCL patients failed to salvage chemotherapy were exposed to dual epigenetic agents (Chidamide 30 mg biw*2w and Decitabine 10 mg/m

Primary testicular lymphoma (PTL) is quite rare. Immunohistochemical analyses show that the majority of cases are diffuse large B-cell lymphoma accounting for about 1–7% of all testicular malignanc...

Acute erythroid leukemia (AEL), characterized by a predominant erythroid proliferation, is a subtype of acute myelogenous leukemia. The genetic basis of AEL remains poorly defined. Through whole-exome sequencing, we identified high frequencies of mutations in CEBPA (32.7%), GATA2 (22.4%), NPM1 (15.5%), SETBP1 (12.1%) and U2AF1 (12.1%). Structure prediction analysis revealed that most of the GATA2 mutations were located at the DNA-binding N-terminal zinc-finger near the DNA-binding interface, suggesting that mutations could result in at least partial inactivation of GATA2 protein. On co-transfection of a GATA-responsive reporter construct together with plasmids expressing either GATA2 wild-type or GATA2 ZF1 mutants (P304H, L321P and R330X) in 293T cells, we found a reduced transcriptional activation in cells transfected with GATA2 mutants. To determine whether reduced GATA2 function is involved in leukemogenesis of AEL, we transfected 32D cells with GATA2 mutants and evaluated the impact of GATA2 mutations on erythroid differentiation. Our data revealed an increased expression of erythroid-related antigens Ter-119, β-globin and βh1-globin, as well as increased hemoglobin positivity in 32D cells transfected with GATA2 mutants compared with control cells. Our results suggest that the decline of GATA2 resulting from mutations contributes to the erythroid commitment, differentiation and the development of AEL.

Background: Chemo-resistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). It has been previously reported that DNA hypermethylation and histone deacetylation are two major epigenetic modifications contributing to chemo-resistance in multiple tumors. Moreover, combination of histone deacetylase inhibitors (HDI) and DNA methyltransferase inhibitors (DNMTI) resulted in synergistic anti-lymphoma effect toward refractory and/or relapsed (R/R) DLBCL cells in vitro and in vivo xenografts. R-GemOx (rituximab, gemcitabine, and oxaliplatin), as a first-line chemotherapy regimen for elderly primary DLBCL patients or a salvage chemotherapy regimen for R/R DLBCL patients not candidates for high-dose therapy, has shown high activity with a relative low toxicity profile. Herein, we therefore aimed to assess the efficacy, safety, and feasibility of the dual epigenetic agents plus R-GemOx regimen (CD-R-GemOx) as a salvage treatment in R/R DLBCL patients. Methods: 13 R/R DLBCL patients including 8 males and 5 females, diagnosed with R/R DLBCL on the basis of the 2008 World Health Organization guidelines, who had failed from previous salvage treatment were exposed to dual epigenetic agents (Chidamide 30mg biw and Decitabine 10mg/m2 qd d1-d5) and sequential R-GemOx(rituximab 375 mg/m² qd d6; gemcitabine 1 g/m² d7,d14; and oxaliplatin 100 mg/m² d8) for salvage chemotherapy. Median age of these patients was 56 (35-67) years old. Most (10/13) patients have advanced Ann Arbor stages. The cycle was repeated every 4 weeks. Clinical efficacy were assessed after two cycles. Results: All thirteen (100%) patients achieved disease control response. Ten (76.9%) patients achieved an overall response at the end of the treatment, with three (23.1%) achieving a complete response. Common grade 3-4 adverse events were haematological toxicities (thrombocytopenia in ten [76.9%] patients, anaemia in six [46.2%], and neutropenia in twelve [92.3%]) and gastrointestinal complications (nausea in four [30.8%] patients, vomiting in one [7.7%], diarrhoea in six [46.2%] and mucositis in six [46.2%]). Besides, 6/13 (46.2%) cases manifested with pyrexia; 3/13(23.8%) cases manifested with increase of ALT and hyperbilirubinemia. All above toxicities were reversible and recovered within 1 month after chemotherapy. No neurological toxicities and treatment-related deaths were observed. Conclusions: Our study indicates that combination of dual epigenetic agents and R-GemOx is a safe and promising salvage approach to managing R/R DLBCL patients and may serve as an optimal salvage treatment for bridging autologous transplantation. Disclosures No relevant conflicts of interest to declare.

Background: Chemoresistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). Chimeric antigen receptor T cells (CAR-T) therapy, as a novel immunotherapy modality, demonstrates impressive efficacy and durable remissions in relapsed/refractory(R/R) B-cell malignancies including DLBCL. However, CAR-T therapy is also associated with potentially fatal toxicities, including cytokine release syndrome (CRS) and neurological toxicities which limit the clinical application of CAR-T therapy in R/R DLBCL with high tumor burden. How to improve the prognosis of this population is urgently underway. Here, we conducted a trial testing efficacy and toxicities of CAR-T therapy in R/R lymphoma patients (This study is registered at www.clinicaltrials.gov as NCT03196830). Methods:Human T cells were collected from autologous/allogenous peripheral blood mononuclear cells (PBMC) . CD3+T cells were separated from PBMS of patients or donors by degradable anti-CD3 magnetic microbeads followed by 24h stimulation with CD3 and CD28 monoclonal antibody (5ug/ml). Then CD3+T cells were transduced with lentivirus particles incorporated with a humanized CAR construct targeting CD19, CD20 and CD22 and further expanded for 7 to 10 days in vitro. The transfection efficacy, ratio of CD4+ versus CD8+, antitumor activities, pathogen detection as well as cytokines releasing of CAR-T cells were evaluated before infusion to patients. In this ongoing trial, 14 patients were diagnosed as R/R DLBCL with high tumor burden. Two debulking conditioning regimens including intensive combined chemotherapy or radiation (40 gray in 20 fractions) were carried out before CAR-T therapy followed by sequential FC regimen (cyclophosphamide 300mg/m2 and fludarabine 30mg/m2 X 3d) for lymphodepletion chemotherapy. Two days after FC regimen, autologous/allogenous CAR-T cells targeting CD19 and CD20 or CD22 provided by the unicar-therapy bio-medicine technology co.(Shanghai, China) at average dose of 1-2×107 cells for each target per kilogram(kg) were infused within 3 days. Results: 14 R/R DLBCL patients with high tumor burden were enrolled in the study. Patients ranged from 39 to 66 years of age had received two to four prior lines of therapy. Out of the 14 patients, 7 patients received intensive second-line chemotherapy (median age, 46 years) and 7 received radiotherapy (median age, 63years) to debulking tumor after leukapheresis and during CAR-T cells preparation. All Adverse Events occurring within 30 days of CAR-T cells infusion were graded and reported for the 14(100%) treated patients. All patients experienced CAR-T-related CRS of any grade with severe CRS (grade 3/4/5) reported in all patients of chemotherapy cohort experienced and mild CRS (grade 1/2) reported in all patients of radiation cohort. 4 patients (57.1%) in chemotherapy cohort manifested with neurological toxicity while no patient in radiation cohort manifested with neurological toxicity. Comparable hematological toxicity as well as non-hematological and non-neurological toxicity were observed in two groups. Four of seven (57.1%) patients achieved an objective response after CAR-T infusion, with three of seven (42.9%) achieving a CR in chemotherapy cohort, while seven of seven (100%) achieved an objective response after CAR-T therapy, with four of seven (57.1%) achieving a CR in radiation cohort. Conclusions:Our clinical trial indicates that compared to intensive second-line chemotherapy, radiation is a better approach to enhancing efficacy and decreasing toxicity of CAR-T therapy in R/R DLBCL patients with high tumor burden. Radiotherapy in combination with CAR-T cell infusion may be the optimal alternative treatment model of R/R DLBCL with high tumor burden. Disclosures No relevant conflicts of interest to declare.

Herein, we describe the establishment and characterization of the first mixed-phenotype acute leukemia cell line (JIH-5). The JIH-5 cell line was established from leukemia cells with B lymphoid/myeloid phenotype from a female mixed-phenotype acute leukemia patient. JIH-5 cells exhibit an immunophenotype comprised of myeloid and B lymphoid antigens. Whole-exome sequencing revealed somatic mutations in nine genes in JIH-5 cells. Transcriptional sequencing of JIH-5 cells identified EP300-ZNF384 fusion transcript, which is a recurrent alteration in B cell acute lymphoblastic leukemia. Our results suggest that the JIH-5 cell line may serve as a tool for the study of mixed-phenotype acute leukemia or EP300-ZNF384.

Among 4,780 consecutive adult acute lymphoblastic/myeloblastic leukemia patients, we identified 117 (2.4%) patients with mixed-phenotype acute leukemia fulfilling WHO 2008 criteria; these were classified as: Blymphoid+ myeloid (n=64), T-lymphoid+myeloid (n=38), B+T-lymphoid (n=14) and trilineage (n=1). Of 92 patients karyotyped, 59 were abnormal and were classified as: complex (22 of 92), t(9;22)(q34;q11) (14 of 92), monosomy 7 (7 of 92), polysomy 21 (7 of 92), t(v;11q23) (4 of 92), t(10;11)(p15;q21) (3 of 92), while STIL-TAL1 fusion was detected in one (T+My) patient. After investigating common acute leukemia-related mutations in 17 genes, 12 of 31 (39%) patients were found to have at least one mutation, classified with: IKZF1 deletion (4 of 31), and EZH2 (3 of 31), ASXL1 (3 of 31), ETV6 (2 of 31), NOTCH1 (1 of 31), and TET2 (1 of 31) mutations. Array-CGH revealed genomic deletions of CDKN2A (4 of 12), IKZF1 (3 of 12), MEF2C (2 of 12), BTG1 (2 of 12), together with BCOR, EBF1, K-RAS, LEF1, MBNL1, PBX3, and RUNX1 (one of 12 each). Our results indicate that mixed-phenotype acute leukemia is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular genetic features.

Acute myeloid leukemia (AML) is a heterogeneous hematological cancer characterized by abnormal growth and a block in myeloid differentiation. The “two-hit” theory suggests that two somatic mutation...

Background Mutations in the PHF6 gene were recently described in patients with T-cell acute lymphoblastic leukemia and in those with acute myeloid leukemia. The present study was designed to determine the prevalence of PHF6 gene alterations in T-cell acute lymphoblastic leukemia. Design and Methods We analyzed the incidence and prognostic value of PHF6 mutations in 96 Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were screened by real-time quantitative polymerase chain reaction and array-based comparative genomic hybridization. Patients were also investigated for NOTCH1 , FBXW7 , WT1 , and JAK1 mutations together with CALM-AF10 , SET-NUP214 , and SIL-TAL1 gene rearrangements. Results PHF6 mutations were identified in 11/59 (18.6%) adult and 2/37 (5.4%) pediatric cases of T-cell acute lymphoblastic leukemia, these incidences being significantly lower than those recently reported. Although PHF6 is X-linked and mutations have been reported to occur almost exclusively in male patients, we found no sex difference in the incidences of PHF6 mutations in Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were detected in 2/79 (2.5%) patients analyzed. NOTCH1 mutations, FBXW7 mutations, WT1 mutations, JAK1 mutations, SIL-TAL1 fusions, SET-NUP214 fusions and CALM-AF10 fusions were present in 44/96 (45.8%), 9/96 (9.4%), 4/96 (4.1%), 3/49 (6.1%), 9/48 (18.8%), 3/48 (6.3%) and 0/48 (0%) of patients, respectively. The molecular genetic markers most frequently associated with PHF6 mutations were NOTCH1 mutations ( P =0.003), SET-NUP214 rearrangements ( P =0.002), and JAK1 mutations ( P =0.005). No differences in disease-free survival and overall survival between T-cell acute lymphoblastic leukemia patients with and without PHF6 mutations were observed in a short-term follow-up. Conclusions Overall, these results indicate that, in T-cell acute lymphoblastic leukemia, PHF6 mutations are a recurrent genetic abnormality associated with mutations of NOTCH1 , JAK1 and rearrangement of SET-NUP214 .

Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Th e BRAF gene is a member of the Raf kinase family and encodes a serine/threonine-specifi c protein kinase that plays an important role in regulating the mitogen activated protein (MAP) kinase/ERK signaling pathway and aff ects cell division, diff erentiation and secretion. Somatic mutations in the BRAF gene occur in a wide variety of human tumors, most commonly in melanoma (43%) and thyroid (39%), colon (12%) and ovarian (12%) cancers. BRAF missense mutations are mainly localized in the glycine-rich loop and activation segment, encoded by exon 11 and exon 15, respectively. A missense mutation in DNA (1799T → A) resulting in an amino acid change from valine to glutamic acid at codon 600 (V600E) accounts for approximately 80% of BRAF mutations found in human cancers [1]. Recently, the BRAF V600E mutation was found in all 48 patients with hairy-cell leukemia (HCL) in one study [2]. Nevertheless, the BRAF V600E mutation has been reported to be rare in other myeloid and lymphoid neoplasms [2–15]. In order to confi rm the prevalence of the BRAF V600E mutation in hematologic malignancies other than HCL, we investigated the prevalence of this gene mutation in patients with a variety of hematologic malignancies. A total of 780 patients were enrolled in this study, including 288 patients with de novo acute myelogenous leukemia (AML), 84 with de novo myelodysplastic syndromes (MDS), 84 with myeloproliferative neoplasms (MPN), 71 with chronic myeloid leukemia (CML) in chronic phase, 51 with CML in blast crisis, 10 pediatric and 102 adult patients with precursor B-cell acute lymphoblastic leukemia (B-ALL), four pediatric and 13 adult patients with T-cell acute lymphoblastic leukemia (T-ALL), 47 with chronic lymphocytic leukemia (CLL) and 26 with multiple myeloma (MM) with bone marrow infi ltration ( 30%). Diagnoses in these patients were made according to the French–American–British (FAB) classifi cation and revised with application of the World Health Organization (WHO) 2008 classifi cation. Th e disease burden for all patients was above the sensitivity of the Sanger direct DNA sequencing technique. Th e median age of the case series was 42 years (range 5–88 years), and the majority were male (54%) and mostly ethnic (Han) Chinese. Th e main characteristics of patients in this study are summarized in Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/10428194.2012. 695777. Th e study was approved by the Ethics Committee of the First Affi liated Hospital of Soochow University and was conducted according to the Declaration of Helsinki. We examined the BRAF V600E mutation by polymerase chain reaction (PCR) amplifi cation of exon 15 of the BRAF gene followed by direct bidirectional DNA sequencing in cells from 780 patients with hematologic malignancies. Genomic DNA was extracted from frozen bone marrow mononuclear cells (BMMCs) collected at diagnosis after Ficoll gradient centrifugation using standard procedures. For PCR amplifi cation of exon 15 of the BRAF gene, primers 5 ’ -TCATAATGCTTGCTCTGATAGGA-3 ’ (forward) and 5 ’ -GGCCAAAAATTTAATCAGTGGA-3 ’ (reverse) were used for both PCR and sequencing. PCR products were directly sequenced on both strands using an ABI 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA). Th e aim of this study was to determine the occurrence of the somatic BRAF V600E mutation in patients with hematologic malignancies, excluding HCL, especially in patients with myeloid malignancies, ALL or MM. We sequenced exon 15 in all 780 patients enrolled in the study. However, no evidence of BRAF mutations invoving exon 15, including the most common mutation BRAF V600E, was found in these patients. By contrast, the BRAF V600E mutation was detected in BMMCs from fi ve patients with HCL. Our data confi rm that the BRAF V600E mutation is common in HCL but indicate it to be absent in MDS, AML, MPN, precursor B-ALL, T-ALL, MM and lymphoma, suggesting that it may not be widespread in hematologic malignancies, excluding HCL.