Humanized CD19 CAR-T cells in relapsed/refractory B-ALL patients who relapsed after or failed murine CD19 CAR-T therapy.

BACKGROUND: For CD19-positive relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) after treatment with murine CD19 (mCD19) CAR-T, the reinfusion of mCD19 CAR-T cells may be ineffective due to anti-mouse single-chain variable fragment (scFv) antibody caused by mCD19 CAR. To overcome this immunogenicity, we applied humanized CD19 (hCD19) CAR-T cells to treat r/r B-ALL patients with prior mCD19 CAR-T therapy. METHODS: Nineteen pediatric and adult patients were included, 16 relapsed after and 3 were primarily resistant to mCD19 CAR-T. All patients presented with more than 5% blasts in bone marrow and/or extramedullary disease, and still showed CD19 antigen expression. Humanized CD19-CARs were lentiviral vectors carrying a second generation CAR with 4-1-BB co-stimulatory and CD3ζ signaling domains. Patient-derived cells were collected for producing CAR-T cells, the median dose of infused hCD19 CAR-T cells was 2.4 × 105/kg (range, 1.0-18.0 × 105/kg). RESULTS: hCD19 CAR-T resulted in a complete remission (CR) rate of 68% (13/19). Among 13 remission patients, 11 underwent allogeneic hematopoietic cell transplantation (allo-HCT) (3 were second HCT) and 10 remained in CR; the event-free survival rates at 12-18 months were 91% in 11 patients received following allo-HCT and 69% in all CR patients. Six cases had no response to hCD19 CAR-T, 3 died of disease progression; another 3 received salvage second transplantation, of them, 2 relapsed again (one died). Cytokine release syndrome (CRS) occurred in 95% (18/19) of patients, most CRS events were grade 1 and grade 2 (n = 17), there was only one grade 4 CRS. Two cases experienced grade 1 neurotoxicity. CONCLUSIONS: Humanized CD19 CAR-T cell therapy could be a treatment option for CD19-positive B-ALL patients who relapsed after or resisted prior murine CD19 CAR-T, hCD19 CAR-T followed by allo-HCT provided a longer remission in CR patients. Nevertheless, the prognosis of non-responders to hCD19 CAR-T remained dismal. TRIAL REGISTRATION: Chinese Clinical Trial Registry/WHO International Clinical Trial Registry ( ChiCTR1900024456 , URL: www.chictr.org.cn ); registered on July 12, 2019.

Combination of CD19 and CD22 CAR-T cell therapy in relapsed B-cell acute lymphoblastic leukemia after allogeneic transplantation.

The prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic transplantation is dismal when treated with conventional approaches. While single-target CD19 or CD22 chimeric antigen receptor (CAR) T-cell therapy has achieved high complete remission (CR) rates in refractory/relapsed B-ALL, it could not maintain a durable remission in most patients. To prolong relapse-free survival, we sequentially combined CD19 and CD22 CAR-T cells to treat post-transplant relapsed B-ALL patients with both CD19/CD22 antigen expression on lymphoblasts. Patient-derived donor cells were collected to produce CAR-T cells that were transfected by lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB. The second T-cell infusion was scheduled at least 1 month, and usually within 6 months after the first CAR-T treatment. Twenty-seven adult and pediatric patients, including 11 (41%) with extramedullary diseases (EMD), received the first CD19 CAR-T and 23 (85%) achieved CR. Subsequently, 21 out of 27 patients received the second CD22 CAR-T and were followed-up for a median of 19.7 (range, 5.6-27.3) months; 14 cases remained in CR, seven relapsed and two of them died from disease progression; Kaplan-Meier survival analysis showed overall survival and event-free survival rates of 88.5% and 67.5%, respectively, at both 12 months and 18 months. CAR-T associated graft-versus-host disease (GVHD) occurred in 23% of patients, with 8% new-onset acute GVHD and 15% persistent or worsened pre-existing cGVHD before CAR-T. This combination strategy of sequential CD19 and CD22 CAR-T therapy significantly improved the long-term survival in B-ALL patients who relapsed after transplantation.

[Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Chinese children: a primary study on clinical characteristics and gene mutations].

OBJECTIVES: To study the clinical features and gene mutations of EBV-HLH in Chinese children. METHODS: Sixteen pediatric patients with Epstein-Barr virus-associated HLH were enrolled the study from May 2011 to September 2012, who were admitted to Capital Institute of Pediatrics. All patients' clinical features were recorded and their nucleotide sequences of all exons and their flanking intronic sequences of perforin (PRF1) gene, protein unc-13 homolog D (UNC13D) gene, two fusion protein genes of syntaxin 11 (STX11)gene, synaptic binding protein 2 (STXBP2) gene, SH2 domain 1A (SH2D1A) gene, and the X-linked inhibitor of apoptosis protein (XIAP) gene were amplified by PCR followed by direct sequencing. Based on HLH gene detection results, all patients divided into the positive and negative gene EBV-HLH subgroups. Statistical analysis was conducted using SPSS 11.5 software. RESULTS: Seven of sixteen pediatric patients with EBV-HLH were identified with heterozygous, hemizygous, and homozygous mutations in PRF1, UNC13D, STXBP2, and SH2D1A. No significant differences were found on the gender, age, illness duration, EBV load, the positive duration of EBV-DNA, lab results, clinical symptoms, treatment, and the outcome between the HLH gene positive and negative subgroups (P > 0.05). CONCLUSIONS: A considerable EBV-HLH pediatric patients have genetic defects, and HLH gene defects may not affect the clinical features and treatment of EBV-HLH pediatric patients.

High doses of mother's lymphocyte infusion to treat EBV-positive T-cell lymphoproliferative disorders in childhood.

Donor lymphocyte infusion is an alternative treatment for Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPDs) but with risk of graft-versus-host diseases (GVHDs). According to the fetal-maternal microchimerism tolerance, we assumed that maternal lymphocyte infusion may be effective without causing GVHD. In 54 cases when a child required cytotherapy or hematopoietic stem cell transplantation, we studied the mother for child-mother microchimerism with use of insertion-deletion polymorphisms as allogeneic markers and a combination of nested polymerase chain reaction (PCR) and real-time quantitative PCR. Thirteen mothers were child-microchimerism-positive at the ratio of 10(-5)-10(-3). Among them, 5 children had non-transplant-associated, EBV(+) T-cell LPD. In these 5 cases, high doses of human leukocyte antigen-haploidentical maternal peripheral blood mononuclear cells (> 10(8)/kg/infusion) were infused 1-4 times. Symptoms of all 5 patients improved between 3 and 10 days after the infusion; thereafter, 3 cases showed complete remission for 6-18 months without further therapy and 2 had partial remission. During the period of observation, none developed obvious GVHD. By quantitative PCR, in some patients maternal cells were found to be eliminated or decreased after infusions, indicating existence of host-versus-graft reaction. We suggest that high doses of mother's lymphocyte infusion may be an effective and safe treatment for non-transplant-associated EBV(+) T-cell LPD.

CAR-T therapy as a consolidation in remission B-ALL patients with poor prognosis.

BACKGROUND: To date, almost all studies regarding chimeric antigen receptor (CAR)-T cell therapy for B-cell acute lymphoblastic leukemia (B-ALL) were performed in refractory/relapsed (r/r) or minimal residual disease-positive patients. CAR-T therapy in remission patients has not been reported. AIM: To observe the treatment outcome of CAR-T cells for remission B-ALL patients with poor prognosis. METHODS AND RESULTS: CAR-T treatment was applied to two B-ALL patients in remission status who had poor prognostic factors and refused transplantation, and one case was unable to accept standard chemotherapy owing to multiple complications. The procedure of CAR-T therapy in these two remission patients was the same as that in r/r B-ALL patients. Lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB were used to produce CAR-T cells. Lymphodepleting agents fludarabine and cyclophosphamide were administered prior to cell infusion. Grade I cytokine release syndrome occurred after each T-cell infusion and there was no neurotoxicity. CAR-T treatment followed by non-intensive maintenance chemotherapy and targeted drugs allowed both patients to obtain a long-term event-free survival of more than three and a half years without transplantation. CONCLUSIONS: CAR-T therapy could be used in high-risk B-ALL patients as a consolidation to avoid transplantation, the combination of CAR-T and following maintenance therapy may be better than CAR-T alone for durable remission.

Case report: Plasma cell leukemia secondary to multiple myeloma successfully treated with anti-BCMA CAR-T cell therapy.

Chimeric antigen receptor (CAR)-T cell therapy provides an effective salvage treatment for relapsed/refractory multiple myeloma (RRMM) patients. End-stage RRMM with plasma cell leukemia (PCL) transformation is highly aggressive and resistant to conventional therapy. There is an urgent need for new therapeutics and CAR-T therapy may play an important role. We report a case of PCL secondary to RRMM successfully treated with CAR-T cell therapy targeting B-cell maturation antigen (BCMA). A woman was diagnosed as having MM 4 years ago and progressed to secondary PCL (sPCL) of five prior lines of treatment including proteasome inhibitors, an immunomodulatory agent, cytotoxic drugs, and an anti-CD38 monoclonal antibody. After receiving a BCMA CAR-T therapy, she achieved a stringent complete response that lasted 9 months. Then, the patient irregularly took venetoclax 10 mg per day due to a slightly higher λ FLC concentration, which did not meet the criteria for progression. She maintained a complete response for the following 7 months. In conclusion, BCMA CAR-T therapy may be a promising therapeutic approach in PCL patients. More studies are needed to evaluate the benefit of anti-BCMA CAR-T therapy in PCL patients. Clinical Trial Registration: www.chictr.org.cn, ChiCTR1900024388, Registered 9 July 2019.

Sequential different B-cell antigen-targeted CAR T-cell therapy for pediatric refractory/relapsed Burkitt lymphoma.

Single antigen-targeted chimeric antigen receptor (CAR) T-cell therapy may be insufficient to induce a durable response in pediatric aggressive B-cell lymphomas. This clinical trial examined the feasibility of sequential different B-cell antigen-targeted CAR T-cell therapy for pediatric relapsed/refractory (R/R) Burkitt lymphoma. Twenty-three patients received the first CD19 CAR T-cell infusion. The patients who did not achieve an ongoing complete response (CR) underwent 1 or more sequential infusions of CAR T-cell therapy that targeted CD22 followed by CD20 according to their disease status and CAR T-cell persistence after each infusion. The median time from the last infusion to the cutoff date was 17 months (range, 15-23 months). The estimated 18-month CR rate was 78% (95% confidence interval [CI], 54%-91%). The estimated 18-month progression-free survival rate was 78% (95% CI, 55%-90%), with 78% (95% CI, 37%-94%) in patients with bulky disease and 60% (95% CI, 25%-83%) in patients with central nervous system (CNS) involvement. During the first CD19 CAR T-cell infusion, grade ≥3 cytokine release syndrome (CRS) occurred in 34.8% and neurotoxicity occurred in 21.7% of all patients. During subsequent infusions, there were only a few incidences of grade >2 CRS and neurotoxicity. All adverse events were reversible. The severity of neurotoxicity was not significantly different between patients with CNS involvement and those who did not have CNS involvement. Sequential CAR T-cell therapy may result in a durable response and is safe in pediatric R/R Burkitt lymphoma. Patients with CNS involvement may benefit from sequential CAR T-cell therapy. This trial was registered at www.chictr.org.cn/index.aspx as #ChiCTR1800014457.

[An analysis of etiological and genetic factors of a patient with familial hemophagocytic lymphohistiocytosis].

OBJECTIVE: To analyze the etiological factor and genetic feature of a familial hemophagocytic lymphohistiocytosis patient with PRF1 mutation (FHL2) with human herpesvirus 7 (HHV7) infection and its family constellation. METHODS: Clinical characteristics, laboratory examinations of a FHL2 case with HHV7 infection were reported. HHV1-HHV8 virus DNA was screened by PCR; NK cell function was analyzed by flow cytometry; PRF1 gene mutations were analyzed by PCR and direct sequencing, structure of mutant PRF1 proteins were analyzed using ExPasy and I-TASSER server and genetics pedigree were analyzed. RESULTS: The patient's HHV7 viral was detected positive with DNA copy number of 350/10(6) peripheral nucleated cells. Flow cytometry analysis showed decrease both in proportion of perforin positive NK cells and perforin protein expression. Genetic testing showed PRF1 biallelic heterozygote mutations (c.503G > A/p.S168N and c.1177T > C/p.C393R) and pedigree analysis showed they were inherited. The patient was then treated with antivirus therapy, dexamethasone and VP16 therapy, but only achieved partial response. The patient was then followed by human leukocyte antigen 10/10 allele identical non-consanguinity allogeneic hematopoietic stem cell transplantations (allo-HSCT) and soon the successful implantation of donor hematopoietic cells and persistent recovery was achieved. The patient was now surviving without recurrence for 9 months after allo-HSCT. CONCLUSIONS: FHL is prone to be misdiagnosed as lymphoma. Genetic analysis of related gene mutation and herpes simplex virus detection will help in early and accurate diagnosis. Allo-HSCT is a fundamental treatment of FHL.

[Monitoring of early Epstein-Barr virus reactivation and preemptive therapy after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To investigate early Epstein-Barr virus (EBV) reactivation and the outcome of preemptive therapy after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: From January 2007 to January 2009, totally 277 patients after allo-HSCT were studied (haploidentical 116, unrelated 75, matched sibling 86). Conditioning regimens were mainly busulfan (BU) + cyclophosphamide (CY)/fludarabine (Flu) or total body irradiation (TBI) + CY/Flu. Antihuman thymocyte globulin (ATG) was added in haploidentical and unrelated transplants. Plasma EBV DNA was monitored once to twice weekly in the first 3 months after allo-HSCT with real time quantitative polymerase chain reaction (RQ-PCR). EBV viremia was diagnosed when EBV DNA was more than 5 × 10(2) copies/ml but without symptoms. Acyclovir (10 mg/kg, intravenous drip, 8 h) was used for preemptive therapy and immuno-suppressants were decreased if possible. RESULTS: Totally 33 patients (11.9%) developed EBV viremia with a median time at day 44 (day 19 to day 84). The incidences of EBV viremia in the transplants from matched sibling, haploidentical, unrelated donors were 0, 15.5%, 20.0%, respectively. There was no significant difference between haploidentical and unrelated transplants (P = 0.09), but much less EBV viremia was seen in matched sibling transplant (P = 0.001). Twenty of 33 patients (60.6%) had complete response to preemptive therapy. The median time to reach EBV DNA negative in plasma was 11 (4 - 56) d. The median duration of preemptive therapy was 21 (14 - 60) d. Both univariate and multivariate analysis indicated that haploidentical and unrelated transplants, acute graft versus host disease (GVHD) were the risk factors for EBV viremia. Two-year overall survival in the patients with EBV viremia was significantly lower than that without EBV viremia (54.2% vs 72.1%, P = 0.006). CONCLUSIONS: Our large clinical study has demonstrated that preemptive therapy with acyclovir that is guided by EBV viremia is effective in majority of the patients with high-risk for EBV reactivation after allo-HSCT, which may further decrease the risk for developing life-threatening EBV disease or post-transplantation lymphoproliferative disorder. Haploidentical and unrelated transplants, acute GVHD are the risk factors for EBV viremia which has negative impact on survival.

[The study of gene mutations in unknown refractory viral infection and primary hemophagocytic lymphohistiocytosis].

OBJECTIVE: To study the type and corresponding clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) associated immune gene mutations in the refractory virus infection or HLH of unknown causes. METHODS: From December 2009 to July 2010, the patients with refractory virus infection or HLH of unknown causes were screened for the primary HLH associated immune genes mutations by DNA sequence analysis, including PRF1, UNC13D, STX11, STXBP2, SH2D1A and XIAP. The clinical characteristics and outcomes were followed up. RESULTS: Totally 25 patients with refractory virus infection or HLH of unknown causes were investigated for the 6 genes and 13 cases were found carrying gene mutations, composing of 6 of PRF1 mutation, 3 of UNC13D, and each one of STX11, XIAP, SH2D1A and STXBP2, respectively. Among the 13 cases with gene mutations, 5 suffered from Epstein-Barr virus associated HLH (EBV-HLH), 1 human herpes virus 7 associated HLH (HHV7-HLH), 1 HLH without causes, 4 chronic activated EB virus infection (CAEBV) with 1 progressing to Hodgkin's lymphoma carrying abnormal chromosome of t(15;17) (q22;q25) and hyperdiploid, 2 EBV associated lymphoma. Among the other 12 patients without gene mutation, 4 suffered from EBV-HLH with 1 progressing to peripheral T lymphoma, 8 suffered from CAEBV. CONCLUSIONS: Primary HLH associated immune gene mutations are critical causes of refractory virus infection of unknown causes, most patients manifest as HLH, some cases appear in CAEBV and EBV associated lymphoma. DNA sequence analysis is helpful to early diagnosis and correct decision-making for treatment.

Peripheral leukemia burden at time of apheresis negatively affects the clinical efficacy of CART19 in refractory or relapsed B-ALL.

Our previous clinical study achieved complete remission (CR) rates of >90% following chimeric antigen receptor T cells targeting CD19 (CART19) treatment of refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL); however, the influence of the leukemia burden in peripheral blood (PB) blasts remains unclear. Here, we retrospectively analyzed 143 patients treated with CART19 (including 36 patients with PB blasts) to evaluate the effect of peripheral leukemia burden at the time of apheresis. One hundred seventeen patients with high disease burdens achieved 91.5% CR or incomplete count recovery CR and 86.3% minimal residual disease-negative CR, and 26 patients with low disease burdens obtained 96.2% MRD- CR. Collectively, 9 of 36 (25%) patients with PB blasts and 2 of 107 (1.87%) patients without PB blasts did not respond to CART19 therapy. The leukemia burden in PB negatively influenced ex vivo cell characteristics, including the transduction efficiency of CD3+ T cells and their fold expansion, and in vivo cell dynamics, including peak CART19 proportion and absolute count, fold expansion, and persistence duration. Further studies showed that these patients had higher programmed death-1 expression in CART19 products. Our data imply that PB blasts negatively affected CART19 production and the clinical efficacy of CART19 therapy in patients with r/r B-ALL.

[The clinical and experimental analyses of a multiple myeloma patient with derivative der(Y;1)].

OBJECTIVE: To investigate the clinical significance of a rare chromosome abnormality der(Y)t(Y;1) in a patient with multiple myeloma (MM). METHODS: The chromosome spread was prepared after 24 h culture of bone marrow. G-banding technique was used to analyze the karyotype. Fluorescence in situ hybridization (FISH) was performed to ascertain the origin of abnormal chromosome detected by conventional karyotypic analysis. Flow cytometry was used to detect the expression of the CD38/CD138/ZAP70. Immunoelectrophore was applied to identify the type of immunoglobulin. RESULTS: A complex pattern of chromosome rearrangement was observed: 92,XXYY[3]/49,X,der(Y)t(Y;1)(q12;q21),t(11;14)(q13;q32),+18,+20,+21[47]/49,X,idem,del(13q22),ace[1]/98,XX,der(Y)t(Y;1) x 2,+18,+18,+20,+20,+21,+21[10]/46,XY[19]. The result was confirmed by metaphase-FISH. The type of immunoglobulin was IgD with the level of 6.24g/L. The CD38/CD138 was positive but ZAP70 was negative. CONCLUSION: Structural abnormality of chromosome Y is rare in blood malignancy. Most of them were described in myelodysplastic syndrome or myeloproliferative disorders. It is the first report of der(Y)t(Y;1) abnormality in multiple myeloma.

Post-HSCT relapsed precursor B-cell ALL successfully salvaged by BiTE followed by consolidation CAR-T cell therapy: A report of two cases.

Precursor B-cell acute lymphoblastic leukemia (pre-B ALL) relapse after allogeneic hematopoietic stem cell transplantation (HSCT) typically has poor outcomes. Both bispecific T-cell engager (BiTE, blinatumomab) and chimeric antigen receptor T-cell (CAR-T) are novel immuno-designed therapies for advanced acute lymphoblastic leukemia. However, the treatment and effects of their combination remain unclear. Two patients with pre-B ALL experienced overt leukemic relapse after allogeneic HSCT. Both patients received one standard cycle of blinatumomab treatment, and complete remission was achieved. Subsequently, during remission, both patients underwent treatment with CAR-T cells prepared from their own T-cells. One patient received CD22-CAR-T cell as a consolidative therapy, while the other underwent CD19-CAR-T cell therapy. No cytokine release syndrome occurred. After treatment, both patients are alive and do not have leukemia recurrence for more than one year. In conclusion, sequential combination of BiTE followed by CAR-T cell therapy may show promising results for relapsed and advanced B-cell leukemia. This novel combination is worthy of further investigation.

[Major molecular response to imatinib in a patient with acute mixed lineage leukemia expressing a novel BCR/ABL transcript].

OBJECTIVE: To identify the novel BCR/ABL transcript in a patient with acute mixed lineage leukemia (AMLL), and to evaluate the imatinib treatment response by quantitatively monitoring the aberrant BCR/ABL. METHODS: Specimens of peripheral blood and bone marrow were obtained. By using several detect protocols, we found a novel BCR/ABL transcript in a patient with Ph-positive AMLL. The patient was treated with Imatinib and the aberrant BCR/ABL was quantitatively monitored to evaluate the clinical response. RESULTS: On admission, cytogenetic analysis showed Philadelphia-chromosome (Ph) positive in the specimens, but BCR/ABL e1a2, b2a2, and b3a2 were negative. Morphological analysis of the bone marrow showed the myeloid blast cells accounted for 66%, and immunophenotyping analysis showed 2 groups of aberrant blast cells: myeloid and B lineage. Chemical therapy and bone marrow transplantation failed to control the disease, and a novel BCR/ABL transcript (GenBank: EF423615) was found by using several detection protocols. The novel fusion protein showed a deletion of 10 amino acids and H893Q compared with the common BCR/ABL b2a2 fusion protein. The patient was then treated with imatinib and hematological remission was soon achieved, and 5 months after the imatinib treatment the quantity of the aberrant BCR/ABL was gradually decreased to negative. During the treatment the patient had discontinued the drug once and just then the aberrant BCR/ABL became positive soon. Imatinib was administered again and molecular remission was soon achieved for the second time. By continued therapy with imatinib, the patient got sustained and complete molecular remission lasting 12 months so far. CONCLUSION: The aberrant BCR/ABL may contribute to the clinical features of AMLL and the AMLL patients that have aberrant BCR/ABL may be sensitive to Imatinib.

CD22 CAR T-cell therapy in refractory or relapsed B acute lymphoblastic leukemia.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

[FIP1L1/PDGFRalpha fusion gene-negative chronic eosinophilic leukemia with t(5;12)(q31;p13): a case report and review of literatures].

OBJECTIVE: To deepen the understanding of chronic eosinophilic leukemia (CEL). METHODS: The course of diagnosis and treatment in a case of FIP1L1/PDGFRalpha fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRalpha fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. RESULT: A sixteen-year-old girl had severe anemia, fever, splenomegaly, thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRalpha was negative. An abnormal clone of T cells expressing CD(3)(-), CD(4)(-), CD(8)(+) was found in peripheral blood and pleural fluid, in which the clonal T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. CONCLUSION: The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRalpha(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD(3)(-), CD(4)(-), CD(8)(+)T-cell abnormality is related to the pathogenesis of CEL.

Impact of clinical factors on outcome of leukemia patients with TLS-ERG fusion gene.

We report the clinical features and outcome of 22 TLS-ERG+ leukemia patients (20 AML and 2 B-ALL). TLS-ERG was tightly associated with extramedullary disease (EMD), complex chromosome abnormalities, and high risk gene mutations including IKZF1, WT1, TET2, NOTCH2, and PHF6. The 6-month leukemia free survival (LFS) with and without EMD was 75% and 83.3% (p = .017). 11/20 AML patients received allogeneic hematopoietic stem cell transplantation (HCT). The 1-year overall survival (OS) in non-HCT and HCT group was 62.5% and 90% (p = .026), but the 6-month LFS in non-HCT and HCT group was 55.6% and 100% (p = .192). The 6-month LFS of patients with complete remission (CR) before HCT versus those with no response (NR) was 67.5% and 0, respectively (p = .034). In conclusion, the leukemia burden before HCT and EMD had negative impact on the outcome of TLS-ERG patients; HCT could prolong OS, but could not overcome the poor prognostic impact of TLS-ERG.

Germline cytotoxic lymphocytes defective mutations in Chinese patients with lymphoma.

Certain patients with lymphoma may harbor mutations in perforin 1 (PRF1), unc-13 homolog D (UNC13D), syntaxin 11 (STX11), STXBP2 (syntaxin binding protein 2) or SH2 domain containing 1A (SH2D1A), which causes functional defects of cytotoxic lymphocytes. Data regarding the association between genetic defects and the development of lymphoma in Chinese patients are limited to date. In the present study, 90 patients with lymphoma were analyzed for UNC13D, PRF1, STXBP2, STX11, SH2D1A and X-linked inhibitor of apoptosis. Mutations were observed in 24 (26.67%) patients; 16 patients exhibited mutations in UNC13D, 7 exhibited PRF1 mutations, and 1 exhibited monoallelic mutation in STX11. UNC13D c.2588G>A/p.G863D mutation was detected in 9 patients (10.00%) and in 4/210 controls (1.90%). This mutation was predicted to be pathogenic and it predominantly existed in the Chinese population. These findings suggest that impaired cytotoxic machinery may represent a predisposing factor for the development of lymphoma. Furthermore, these data describe a distinct mutation spectrum in Chinese patients with lymphoma, whereby UNC13D is the most frequently mutated gene. In addition, these findings suggest UNC13D c.2588G>A mutation is a founder mutation in Chinese patients.