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Endometrial cancer, a type of primary epithelial malignant tumor in the endometrium, is one of the three most common malignant tumors of the female reproductive system. While the incidence of endometrial cancer has been recently rising, its etiology remains unclear. In this study we found that EM2D9, an independently developed monoclonal antibody, specifically recognized endometrial cancer cells; we further determined that EM2D9 target protein was α5β1. In vitro and in vivo experiments showed that EM2D9 inhibited the migration of endometrial cancer cells. Real-time quantitative PCR results showed that the expression of CD151 mRNA in endometrial carcinoma cells significantly decreased after EM2D9 treatment. We also found that EM2D9 affected the FAK signaling pathway. Collectively, these results shed light on a new mechanism for the development of endometrial carcinoma.

HIV infection induces a robust Tcell response that is sustained by high viremia, but falls following the onset of antiretroviral therapy (ART). Relatively little has been reported on the subsequent stability of the HIV-specific Tcell response in individuals on durable therapy. Such data are critical for powering clinical trials testing Tcell-based immunotherapies. In a cross-sectional study, HIV-specific Tcell responses were detectable by exvivo interferon (IFN)-γ ELISpot (average ∼1,100 spot-forming units [SFUs]/106 peripheral blood mononuclear cells) in persons living with HIV (PLWH; n= 34), despite median durableART suppression of 5.0 years. No substantial association was detected between the summed HIV-specific Tcell response and the size of the replication-competent HIV reservoir. Tcell responses were next measured in participants sampled weekly, monthly, or yearly. HIV-specific Tcell responses were highly stable over the time periods examined; within-individual variation ranged from 16% coefficient of variation (CV) for weekly to 27% CV for yearly sampling. These data were used to generate power calculations for future immunotherapy studies. The stability of the HIV-specific Tcell response in suppressed PLWH will enable powered studies of small sizes (e.g., n= 6-12), facilitating rapid and iterative testing for Tcell-based immunotherapies against HIV.

Purpose Radical prostatectomy (RP) is a common treatment for prostate cancer, but a fraction of patients may experience PSA recurrence after surgery, manifesting as an elevation in prostate specific antigen (PSA). Vast literature has reported different prognostic factors for PSA recurrence without reaching a consensus. This retrospective study investigated the efficacy of a new indicator in predicting PSA recurrence in patients after RP. Patients and methods From October 2000 to December 2015, 102 PCa patients who underwent laparoscopic prostatectomy in the Urology Department of Peking Union Medical College Hospital were analyzed. We calculated PSApostd3/PSApre, defined as the ratio of the PSA on day 3 postop as the numerator and the pre-operative PSA as the denominator, in these patients to represent PSA decrement after surgery, and investigated its relationship with PSA recurrence during follow-up. Results The receiver operating characteristic (ROC) curve of PSApostd3/PSApre derived a cut-off at 0.453 (sensitivity=0.704, specificity=0.853, P

Background. Diabetic endotoxemia has been recognized as one of the hallmarks of type 2 diabetes mellitus (T2DM). Recent findings suggest that gut leak plays a pivotal role in diabetic endotoxemia. Cortex Mori (CM) has been widely applied in China to ameliorate development of T2DM, but its effect on endotoxemia is unknown. Methods. The study was constructed with two parts: (1) in vivo study of CM on diabetic endotoxemia in db/db mice. Eight C57BL/6 mice were set as normal control; (2) in vitro study of mulberroside A (MBA) from CM on diabetic endotoxemia. Potential mechanism of MBA on ameliorating diabetic endotoxemia was also explored. Results. The present study found that CM water extract decreased levels of blood glucose, ameliorated liver and renal damage in db/db mice, and ameliorated diabetic endotoxemia ( p < 0.01 ). We also found that the water extract enhanced gut integrity and decreased gut inflammatory protein ICAM-1 expression in db/db mice as detected by H&E staining and immunohistochemistry methods. In the in vitro study, MBA decreased levels of MDA and ROS induced by LPS ( p < 0.01 ) and enhanced the integrity of gut epithelial barrier ( p < 0.01 ). Conclusions. We found that Cortex Mori and its active component mulberroside A could ameliorate diabetic endotoxemia by preserving gut integrity.

Neurocan (NCAN), a secreted chondroitin sulfate proteoglycan, is one of the major inhibitory molecules for axon regeneration in nervous injury. However, its role in cancer is not clear. Here we observed that high NCAN expression was closely associated with the unfavorable outcome of neuroblastoma (NB). NCAN was also highly and ubiquitously expressed in the early lesions and terminal tumor of TH-MYCN mice, a NB model. Interestingly, exogenous NCAN (i.e., overexpression, recombinant protein and conditioned medium) transformed adherent NB cells into spheres whose malignancies in vitro (anchorage-independent growth and chemoresistance) and in vivo (xenograft tumor growth) were potentiated. Both chondroitin sulfate sugar chains and NCAN's core protein were essential for the sphere formation. The CSG3 domain was essential in the moiety of NCAN. Our comprehensive microarray analysis and RT-qPCR of mRNA expression suggested that NCAN treatment promoted cell division, and urged cells to undifferentiated state. The knockdown of NCAN in tumor sphere cells cultured from TH-MYCN mice resulted in growth suppression in vitro and in vivo. Our findings suggest that NCAN, which stimulates NB cells to promote malignant phenotypes, is an extracellular molecule providing a growth advantage to cancer cells.

To compare the accuracy of magnetic resonance-guided prostate biopsy (MR-GPB) and template-guided transperineal prostate saturation biopsy (TTPSB). A total of 219 patients with elevated prostate-specific antigen, abnormal digital rectal examination or ultrasound findings were enrolled. All patients underwent multiparametric magnetic resonance image (mpMRI). Patients with a Prostate Imaging Reporting and Data System (PI-RADS) score of 3 to 5 underwent MR-GPB using 2 to 5 biopsy cores and then immediately underwent an 11-region TTPSB. Patients with a PI-RADS score of 1 to 2 underwent TTPSB alone. We compared the detection rates for any cancer, clinically significant prostate cancer (csPCA), and the spatial distribution of missed csPCA lesions. Among the 219 cases, 66 (30.1%) had a PI-RADS score of 1 to 2 on mpMRI. The detection rate of TTPSB in these patients was 9.1% (6/66). In total, detection rates for any cancer and csPCA were 48.9% (107/219) and 42.9% (94/219), respectively. Detection rates for any cancer (TTPSB 87/219, 39.7%; MR-GPB76/219, 34.7%, P = .161) and csPCA (TTPSB 76/219, 34.7%; MR-GPB 72/219, 32.9%, P = .636) did not significantly differ between the 2 groups. The csPCA lesions missed by MR-GPB were most commonly located on the left (8.5%, 8/94) and right (9.6%, 9/94) sides of the urethra. MR-GPB can reduce the rate of unnecessary prostate biopsies by approximately 30% and exhibits an efficacy comparable to TTPSB for the detection of any cancer and csPCA. Nevertheless, approximately 1/4 of csPCAs were missed by MR-GPB and were most commonly located on both sides of the urethra.

Background Immunosuppressive therapy (IST) is commonly used for patients with acquired severe aplastic anemia (SAA). Because the clinical response rate and therapeutic outcome for individual patients to IST varies, an in vitro test that identifies potential responders would be desirable. Methods We evaluated the relationship between thrombopoietin (TPO) levels at the time of diagnosis and the response to IST at 6 months in 85 children (median age, 9.0 years; range, 1.0–15.5 years) with acquired SAA using enzyme-linked immunosorbent assay. Thirty-one age-matched healthy individuals were used as controls. All patients received antithymocyte globulin and cyclosporine. Results Overall, 39 patients (45.9%) responded to IST at 6 months. TPO plasma levels were significantly higher in nonresponders than in responders (1,555.8 vs. 1,284.7 pg/ml, respectively; P = 0.031). Multivariate analysis identified the TPO levels of >1,796.7 pg/ml (TPO-high group, 20 patients; odds ratio (OR), 8.285; 95% confidence interval (CI), 2.114–32.904; P = 0.002) as independent poor predictors of IST response at 6 months. Moreover, the TPO-high group was associated with lower 5-year failure-free survival rates (30% vs. 68%, P = 0.012) compared with the TPO-low group. Conclusion The measurement of TPO levels at diagnosis is useful for predicting the response to IST in children with SAA and may help in decision making.

Background Distinguishing hypocellular refractory cytopenia of childhood (RCC) from aplastic anemia (AA) is challenging. Thus far, no studies have compared the cytokine profiles in patients with AA to those with hypocellular RCC. In the present study, we addressed whether thrombopoietin (TPO) and interleukin 17 (IL-17) plasma levels are useful for differentiating between the two diseases. Methods We measured the endogenous plasma concentrations of TPO and IL-17 in 29 patients with AA, 34 patients with hypocellular RCC, and 31 healthy controls using sensitive enzyme-linked immunosorbent assays. Results The TPO and IL-17 plasma levels were significantly lower in patients with hypocellular RCC than in patients with AA (P < 0.001 and P = 0.007, respectively). The multivariate logistic regression analysis identified moderate disease severity, TPO levels of

Liver cancer is the sixth most prevalent cancer, and the third most frequent cause of cancer-related deaths. Circular RNAs (circRNAs), a kind of special endogenous ncRNAs, have been coming back to the forefront of cancer genomics research. In this study, we used a systems biology approach to construct and analyze the circRNA molecular regulatory networks in the context of liver cancer. We detected a total of 127 differentially expressed circRNAs and 3,235 differentially expressed mRNAs. We selected the top-5 upregulated circRNAs to construct a circRNA-miRNA-mRNA network. We enriched the pathways and gene ontology items and determined their participation in cancer-related pathways such as p53 signaling pathway and pathways involved in angiogenesis and cell cycle. Quantitative real-time PCR was performed to verify the top-five circRNAs. ROC analysis showed circZFR, circFUT8, circIPO11 could significantly distinguish the cancer samples, with an AUC of 0.7069, 0.7575, and 0.7103, respectively. Our results suggest the circRNA-miRNA-mRNA network may help us further understand the molecular mechanisms of tumor progression in liver cancer, and reveal novel biomarkers and therapeutic targets.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.

Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, < 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P Conclusions: The current study indicates that NK cells play a role in the pathogenesis of AA and may be the predictors of response to IST. Recovery of NK cell count correlated with hematopoietic recovery following IST, indicating that they may be used as markers to assess disease activity, treatment response and relapse. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

Precise genetic diagnosis of inherited bone marrow failure syndromes (IBMFS), a heterogeneous group of genetic disorders, is challenging but essential for precise clinical decision making. We analyzed 121 IBMFS patients using a targeted sequencing covering 184 associated genes and 250 IBMFS patients using whole-exome sequencing (WES). We achieved successful genetic diagnoses for 53 of 121 patients (44%) using targeted sequencing and for 68 of 250 patients (27%) using WES. In the majority of cases (targeted sequencing: 45/53, 85%; WES: 63/68, 93%), the detected variants were concordant with, and therefore supported, the clinical diagnoses. However, in the remaining 13 cases (8 patients by target sequencing and 5 patients by WES), the clinical diagnoses were incompatible with the detected variants. Our approach utilizing targeted sequencing and WES achieved satisfactory diagnostic rates and supported the efficacy of massive parallel sequencing as a diagnostic tool for IBMFS. Genet Med advance online publication 19 January 2017

Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1–seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1–seronegative patients ( P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1–specific CD4 + and CD8 + T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1–seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1–seropositive patients with associated CD8 + T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8 + T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage ( P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer.

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells and fatty replacement in the bone marrow. Transcriptional factor GATA-2 plays several important roles in both hematopoiesis and adipogenesis. Decreased levels of GATA-2 compromise the proliferation and survival of hematopoietic stem cells. GATA-2 suppresses adipocyte differentiation through direct inhibition of adipogenic factors, including peroxisome proliferator-activated receptor-gamma (PPARgamma). Previous studies have shown that expression of GATA-2 is decreased in marrow CD34-positive cells in AA. To elucidate the mechanisms of fatty marrow replacement, we evaluated the mRNA expression for GATA-2 and PPARgamma in mesenchymal stem cells (MSCs) from patients with AA by quantitative real-time polymerase chain reaction. GATA-2 expression by MSCs from AA patients was significantly lower than in normal subjects. Conversely, expression of PPARgamma was significantly higher in AA patients. Western blot analysis demonstrated that protein levels of GATA-2 were lower in AA patients than those in normal subjects. Moreover, incubation with interferon-gamma induced downregulation of GATA-2 levels in MSCs from normal subjects. These findings indicate that fatty marrow replacement in AA patients can be explained by downregulation of GATA-2 and overexpression of PPARgamma in MSCs. Decreased expression of GATA-2 might be responsible for the pathogenesis and development of the clinical features of the disease.

Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

As a member of the corticotropin-releasing factor (CRF) family, urocortin (UCN) has been demonstrated to show diverse effects on cardiovascular system. It is commonly considered as a protective agent on vascular function. However, a growing body of evidence suggests that some effects of UCN may lead to endothelial dysfunction (ED) which may be the cause or consequence of vascular disease and a hallmark of known cardiovascular risk factors. The present review is then focused on recent evidence that reveals the beneficial and detrimental effects of UCN on endothelium.

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was -0.99 standard deviation (SD) (range -4.01-+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P

We analyzed the correlation between rabbit ATG (rATG) serum levels and clinical outcomes in 37 children who received rATG at a total dose of 10 or 15 mg/kg during HSCT conditioning from an alternative donor. Fourteen patients had advanced malignant diseases, 13 had severe AA, and 10 had inherited disorders. Complete engraftment was achieved in all patients, and no rejection occurred. The cumulative incidence of grades II-IV acute GVHD and extensive chronic GVHD was 27% (95% CI, 12.5-39.6%) and 8.1% (95% CI, 0-23.1%), respectively. Multivariate analysis identified lower rATG levels at week 4 as an independent risk factor in the development of grades II-IV acute GVHD (p = 0.037). Serious infections were not observed in any patient following HSCT. No correlation was found between EBV reactivation and rATG levels at week 2 and week 4 after HSCT. Furthermore, no correlation was found between relapse and rATG levels two and four wk post-transplantation. The probability of five-yr OS among patients was 70.3% (95% CI, 59.8-79.2%). Our results suggest that targeted rATG administration may protect patients from severe acute GVHD without increasing the risk of EBV reactivation or relapse.

GATA2 , a member of the GATA transcription factor family, plays a critical role in hematopoiesis,[1][1] vascular[2][2] and neural development. Mutations in the exons and intron 5 of this gene have been identified as the cause of several hematologic disorders.[3][3] GATA2 -related disorders include

PTPN11, the gene which encodes protein tyrosine phosphatase SHP-2, plays an important role in regulating intracellular signaling. Germline mutations in PTPN11 were first observed in Noonan syndrome, while somatic mutations were identified in hematological myeloid malignancies. Recently, PTPN11 mutations have been reported in children with acute lymphoblastic leukemia (ALL). In the present study, we investigated the prevalence of mutations in PTPN11, RAS and FLT3 in samples from 95 Japanese children with ALL. We observed exon 3 and 8 missense mutations of PTPN11 in 6 children with B precursor ALL. One patient with Down syndrome and ALL had PTPN11 mutation. We also identified RAS mutations in ten patients and FLT3 internal tandem duplication (FLT3/ITD) in one patient. None of the patients had simultaneous mutations in PTPN11 and RAS, while one patient had both PTPN11 and FLT3 mutations. These data suggest that PTPN11 mutation may play an important role for leukemogenesis in a proportion of children with ALL, particularly B precursor ALL.

Predicting the response to immunosuppressive therapy could provide useful information to help the clinician define treatment strategies for patients with aplastic anemia. In our current study, we evaluated the relationship between telomere length of lymphocytes at diagnosis and the response to immunosuppressive therapy in 64 children with aplastic anemia, using flow fluorescence in situ hybridization. Median age of patients was ten years (range 1.5-16.2 years). Severity of the disease was classified as very severe in 23, severe in 21, and moderate in 20 patients. All patients were enrolled in multicenter studies using antithymocyte globulin and cyclosporine. The response rate to immunosuppressive therapy at six months was 52% (33 of 64). The probability of 5-year failure-free survival and overall survival were 56% (95% confidence interval (CI): 41-69%) and 97% (95%CI: 87-99%), respectively. Median telomere length in responders was -0.4 standard deviation (SD) (-2.7 to +3.0 SD) and -1.5 SD (-4.0 to +1.6 (SD)) in non-responders (P

Juvenile myelomonocytic leukemia (JMML) is a mixed myeloproliferative and myelodysplastic disorder that occurs in early childhood. The clinical course of JMML is highly variable. A third of patients follow a relatively indolent course, although approximately 15 % cases are thought to develop acute myeloid leukemia, referred to as blast crisis. The etiology and clinical characteristics of blast crisis remain unclear. We document the case of a 27-month-old boy who presented with hepatosplenomegaly, skin rash, and lymphadenopathy. An initial diagnosis of acute erythroid leukemia was made according to the French–American–British classification. Following estimation of hypersensitivity to GM-CSF and genetic analysis of PTPN11, he was diagnosed with JMML in the blast crisis phase. Although he had several poor prognostic factors, including monosomy 7 and high HbF percentage, he achieved partial remission after treatment with acute myeloid leukemia-oriented chemotherapy followed by allogeneic hematopoietic stem cell transplantation. He has been in complete remission for over 6 years.

10568Background: The primary objective of this study was to assess safety and toxicity of anti-GD2 antibody ch14.18/CHO continuous long term infusion (LTI) used as single agent in Japanese pediatri...

The aim of this study was to investigate changes in CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) throughout the clinical course of Kawasaki disease (KD) and correlations with response to intravenous immunoglobulin (IVIg) therapy. Participants comprised 18 patients who fulfilled the diagnostic criteria for KD and 20 healthy subjects. Expressions of CD25 and FOXP3 among all CD4(+) T cells in peripheral blood mononuclear cells were analyzed by flow cytometry before and 7 and 30 days after IVIg therapy. Before treatment, percentages of CD4(+)CD25(+)FOXP3(+) Tregs among total CD4(+) Tregs were significantly lower among KD patients (4.19 %; range, 0.16-8.11 %) than among healthy subjects (7.32 %; 4.18-13.42 %; P = 0.0001). Both percentages and absolute numbers of CD4(+)CD25(+)FOXP3(+) Tregs on day 7 after IVIg therapy were significantly increased compared with values before treatment (8.02 % (range, 0.51-12.6 %) vs. 4.19 % (range, 0.16-8.11 %), P = 0.0005; 93.25/ μL (range, 6.67-258.05) vs. 41.85/ μL (range, 0.44-160.62), P 0.0001, respectively). Moreover, percentages and absolute numbers of CD4(+)CD25(+)FOXP3(+) Tregs before treatment were significantly lower in the IVIg-resistant group than in the IVIg-sensitive group (0.18 % (range, 0.16-3.34 %) vs. 4.52 % (range, 2.8-8.11 %), P = 0.0022; 0.68/μL (range, 0.44-53.81) vs. 51.66/μL (range, 2.88-160.62), P = 0.0098, respectively). The frequency of CD4(+)CD25(+)FOXP3(+) Tregs in four of the five IVIg-resistant patients at diagnosis was more than 3 standard deviations below that in healthy subjects. Two of these four patients displayed coronary abnormalities, and one of these two patients developed coronary aneurysm.Lack of CD4(+)CD25(+)FOXP3(+) Tregs before treatment may predict resistance to IVIg therapy in patients with KD.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially "boosting" the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

Juvenile myelomonocytic leukemia (JMML) progenitor cells exhibit in vitro hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Phospho-specific flow cytometry using anti-phosphorylated STAT5 antibody is a new method recently reported to detect GM-CSF hypersensitivity of cells. However, colony assays using methylcellulose medium to measure GM-CSF-hypersensitivity remain as the current gold standard. Interestingly, cytomegalovirus (CMV) infection in infancy often presents with a variety of clinical symptoms that mimic JMML, with CMV giving a positive result by colony assay. We wanted to determine whether aberrant STAT5 activation occurs in CMV infection by using phospho-specific flow cytometry, and to ascertain whether this method is effective at discriminating CMV infection from JMML. Peripheral blood mononuclear cells from patients with JMML and CMV infection displayed an elevated proportion of p-STAT5 cells after low-dose GM-CSF stimulation when compared with cells from normal individuals. However, we found no significant differences in the percentage of p-STAT5 positive cells from patients with CMV infection and JMML at any doses of the GM-CSF doses used. We conclude that patients with CMV infection cannot be discriminated from patients with JMML by this new diagnostic method.

Summary Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML-related mutations.

Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines.We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a.Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method.Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.

We describe a 4-month-old infant girl with congenital erythroid and myeloid hypoplasia who developed myelodysplastic syndrome. Bone marrow examination showed severe erythroid and myeloid hypoplasia without dysplastic morphology. Flow cytometry detected autoantibodies to myeloid cells, indicating a diagnosis of Diamond-Blackfan anemia with autoimmune neutropenia. The patient was administered prednisolone and rituximab, which brought the neutrophil count and hemoglobin level to within the normal range. However, bicytopenia recurred at the age of 22 months. She was diagnosed with myelodysplastic syndrome because of trilineage dysplasia and the clonal abnormality of 46,XX,dup(1)(q21;q32) in bone marrow. She was transplanted with cord blood from an unrelated human leukocyte antigen-matched donor and has since remained in complete remission for 20 months.

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs), resulting in peripheral pancytopenia. Several pathogeneses for AA have been hypothesized, and among these, the autoimmune-associated hypothesis is considered to be the most likely [1, 2]. Mesenchymal stem cells (MSCs) have been shown to exert immunosuppressive capacity both in vitro and in vivo. Normal bone marrow (BM)-derived MSCs suppress T cell function and may provide an immunoprotected environment for HSCs [3, 4]. Bacigalupo et al. [5] reported that MSCs derived from the BM of adult AA patients are defective in their ability to suppress T cell proliferation and cytokine release. They hypothesized that these defects may result in increased activation/proliferation of BM T cells, which suppress HSCs in AA patients. The aim of the present study was to investigate whether MSCs from pediatric AA patients also exert decreased suppressive activity on T cells when compared with those derived from normal individuals. Bone marrow samples from 34 AA children (16 males, 18 females) and 15 healthy volunteers were obtained following informed consent from either patients or parents. Median patient age was 10 years (range 2–21 years). The timing of sampling was at diagnosis in 15 patients and after immunosuppressive therapy (IST) with anti-thymocyte globulin and cycrosporin A in 19 patients. Eleven of these were IST responders and eight were non-responders. Aplasia was secondary to acute hepatitis in four patients and the cause was unknown in 30 patients. Seven patients had very severe AA, 19 patients had severe AA and eight patients had moderate AA. Bone marrow mononuclear cells (MNCs) were isolated and cultured as reported previously [5]. Briefly, MNCs were plated in a 25-cm tissue culture flask with complete MesenCult Basal Medium (Stem cell technologies, Vancouver, Canada) containing human MSC supplements. Adherent cells were expanded to the third generation. Characterization of MSCs was determined by flow cytometry and their differentiation into adipogenic and osteoblastic lineages. Surface markers of the MSCs were CD29?, CD44?, CD90?, CD105?, CD73?, CD34-, CD45and CD14-. To demonstrate capacity of MSCs to differentiate to adipocytes, cells were further cultured at 2 9 10 cells/flask with MesenCult basal medium supplemented with human adipocytogenic supplements (Stem cell technologies). Adipogenic differentiation was confirmed by the presence of lipid droplets under optical microscopy after 3 weeks. Similarly, for differentiation to osteoblasts, MSCs were cultured with MesenCult basal medium supplemented with an osteogenic differentiation kit (Stem cell technologies) containing dexamethasone (0.1 lmol/l), ascorbic acid (0.05 mmol/l) and glycerophosphate (10 mmol/l). After 5 weeks of culture, cell layers were stained with silver nitrate (Von Kossa method) in order to detect calcium deposition, which indicates osteogenesis. There were no differences in the expression of MSC surface markers and the differentiation capacity between AA patients and normal individuals. Immunosuppressive activity of MSCs from pediatric patients with AA was tested by their inhibitory effects on PHA-induced T cell proliferation. Peripheral blood MNCs from normal adult volunteers (2 9 10) were dispensed Y. Xu Y. Takahashi A. Yoshimi M. Tanaka H. Yagasaki S. Kojima (&) Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: kojimas@med.nagoya-u.ac.jp

Juvenile myelomonocytic leukemia (JMML), an overlap of myelodysplastic / myeloproliferative neoplasm, is an intractable pediatric myeloid neoplasm. Epigenetic regulation of transcription, particularly by CpG methylation, plays an important role in tumor progression, mainly by repressing tumor-suppressor genes. To clarify the clinical importance of aberrant DNA methylation, we studied the hypermethylation status of 16 target genes in the genomes of 92 patients with JMML by bisulfite conversion and the pryosequencing technique. Among 16 candidate genes, BMP4, CALCA, CDKN2A, and RARB exhibited significant hypermethylation in 72% (67/92) of patients. Based on the number of hypermethylated genes, patients were stratified into three cohorts based on an aberrant methylation score (AMS) of 0, 1–2, or 3–4. In the AMS 0 cohort, the 5-year overall survival (OS) and transplantation-free survival (TFS) were good (69% and 76%, respectively). In the AMS 1–2 cohort, the 5-year OS was comparable to that in the AMS 0 cohort (68%), whereas TFS was poor (6%). In the AMS 3–4 cohort, 5-year OS and TFS were markedly low (8% and 0%, respectively). Epigenetic analysis provides helpful information for clinicians to select treatment strategies for patients with JMML. For patients with AMS 3–4 in whom hematopoietic stem cell transplantation does not improve the prognosis, alternative therapies, including DNA methyltransferase inhibitors and new molecular-targeting agents, should be established as treatment options.

Introduction: Acquired aplastic anemia (AA) is an immune mediated disease characterized by severe quantitative defects in stem cell number resulting in a hypocellular marrow and peripheral blood cytopenias. Immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine A (CyA) is the standard therapy for patients with AA who lack a human leukocyte antigen (HLA)-matched sibling donor. Previous studies identified minor paroxysmal nocturnal hemoglobinuria (PNH) clones and short telomere length (TL) as predictive markers of the response to IST. However, the likelihood ratio of these markers was insufficient to influence clinical decisions. Here we assessed whether the combination of these markers could more accurately predict the response to IST. Methods: Between July 2001 and November 2013, 116 patients (65 boys and 51 girls) diagnosed with AA were examined to identify a minor population of PNH-type cells and TL of peripheral blood lymphocytes. Fifty-three patients received horse ATG (Lymphoglobulin, 15mg/kg/day for 5 days) and 63 received rabbit ATG (Thymoglobulin, 2.5−3.75 mg/kg/day for 5 days). CyA (6mg/kg/day) was started on day 1 and continued to at least day 180. The dose of CyA was adjusted to maintain trough levels between 100 and 200 ng/ml. Written informed consent was obtained from all patients’ parents. The ethics committee of each participating hospital approved the study. We used a flow cytometric assay to detect PNH-type granulocytes and red blood cells (RBCs). Presence of >0.007% CD13+ CD55− CD59− granulocytes and/or >0.009% glycophorin A+ CD55− CD59−RBCs was defined as PNH positive (PNH+), based on the results from healthy individuals. The average relative TL of peripheral lymphocytes was measured by flow-fluorescence in situ hybridization (flow-FISH) using a Telomere PNA Kit (Dako Cytomation). Based on our previous study, we defined −1.0 SD of age-adjusted controls as the cut-off value. Results: The median age at IST was 9.8 years (0.9−16.0). Disease severity of patients was assessed as moderate in 39, severe in 37, and very severe in 40, respectively. The causes of AA were idiopathic in 104 patients and hepatitis-associated in 12 patients. The median follow-up period from the time of IST was 36 months (range 1−134). Thirty-nine patients (34%) carried a minor PNH clone at diagnosis. Compared with healthy individuals, their median of TL was −1.08 SD (−4.01 to +3.01 SD). Overall, 60 of 116 patients (51.7%) responded to IST at 6 months after administration of ATG. In univariate analysis, the response rate at 6 months was higher in PNH+ patients than in PNH- patients (74.4% vs. 40.3%, respectively, p = 0.001). Longer TLs (≥−1.0 SD of age-adjusted control) were also associated with a favorable response to IST at 6 months (73.2% vs. 31.7%, p Conclusion: Combination of the absence of a minor PNH clone and a short TL was an efficient predictor of poor response to IST. These patients had low chances to respond to IST, which challenges the current standard of treatment. Prospective clinical trials are required to confirm these findings. Disclosures No relevant conflicts of interest to declare.

Introduction: The appropriate classification of bone marrow (BM) failure syndromes in children is challenging, particularly in relation to histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study is to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 children with three diseases using whole-exome sequencing. Patients and Methods: Wediagnosed AA, RCC, or RCMD on the basis of morphology and histological findings of bone marrow (BM) according to the 2008 World Health Organization (WHO) classification criteria. Patients with AA exhibited hypocellular BM and no morphologically dysplastic changes in any of three hematopoietic cell lineages, while patients with RCC had 10% in one cell lineage. Patients with RCMD exhibited >10% dysplastic changes in two or more cell lineages. We obtained peripheral blood and BM samples from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). We performed exome capture from paired DNA (non-T cells/CD3+ lymphocytes) using SureSelect® Human All Exon V4 kit (Agilent Technologies, Santa Clara, CA), which covered all part of the coding exons, followed by massively-parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations and germline variants were detected through our pipeline for whole-exome sequencing (Genomon-exome). All candidate somatic nucleotide changes were validated by Sanger sequencing. The ethics committee of Nagoya University Graduate School of Medicine approved this study. Results: Whole-exome sequencing pipeline identified a total of 14 non-synonymous somatic (one nonsense, 11 missense, and two frameshift) changes among the 29 patients, which resulted in only 0.48 mutations per patient. The average numbers of somatic mutations per sample were not significantly different among these groups (0.50 in AA, 0.36 in RCC, and 0.60 in RCMD). As a whole, childhood AA, RCC, and RCMD were characterized by a paucity of somatic mutations compared with adult myelodysplastic syndromes (MDS) in which 10 or more mutations per exome were detected on average. Among the mutated genes, BCOR-inactivating mutations in two patients (p.S158fs in AA and p.E1286X in RCMD) were considered significant genetic events based on previous reports that it is a driver gene in MDS. With regard to germline events, we did not detect any germline mutations of inherited BM failure syndromes. Moreover, we did not identifiy significantly frequent germline events in the entire cohort or any genetic hallmarks to be able to discriminate between these three diseases. When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0%) vs. 6% (0%–26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100%–100%) vs. 95% (70%–99%)] were not significantly different. Conclusion: We usedwhole-exome sequencing analysis for gene mutational profiling of children with AA, RCC, and RCMD. Idiopathic bone marrow failure syndromes in children are characterized by a paucity of somatic gene mutations, irrespective of histological diagnosis. These findings suggest that histological diagnosis based on the WHO classification system does not discriminate the mutational profile of idiopathic BM failure syndromes in children. Disclosures No relevant conflicts of interest to declare.

Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests. Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies. Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes. Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted. Disclosures No relevant conflicts of interest to declare.

Recent studies suggest that aberrant methylation plays a fundamental role in the development of a variety of cancers, including myeloid malignancies. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is characterized by both excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor. It is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) according to the World Health Organization classification. We recently reported that somatic mutations in SETBP1 and JAK3 were identified in JMML patients and were associated with poor outcomes (Nat Genet 2013;45:937–41). The goal of this study was to clarify the clinical significance of aberrant DNA methylation in JMML. We studied 92 children (61 boys and 31 girls) who were diagnosed with JMML in institutions throughout Japan. A diagnosis of JMML was made based on internationally accepted criteria. We quantitatively evaluated the CpG methylation pattern in the promoter regions of 16 candidate genes (APC, BMP4, CALCA, CDH13, CDKN2A, CDKN2B, CHFR, DAPK, DMR-H19, ER, IGF2, MGMT, MLH1, RARB, RASSF1, TP73) from genomic DNA derived from bone marrow specimens at the time of diagnosis. This was accomplished by bisulfite conversion and the pryosequencing technique. We defined aberrant methylation as >3 standard deviations from the mean methylation level derived from 8 healthy individuals. The median age at diagnosis was 16 months (range, 0.3–160). By genetic analysis, PTPN11, NF1, NRAS, KRAS, and CBL mutations were found in 39 (42%), 7 (8%), 12 (13%), 13 (14%), and 11 (12%) patients, respectively. In addition, 16 patients had SETBP1 or JAK3 mutations. Karyotypic abnormalities were detected in 15 patients, including 8 with monosomy 7. The median monocyte count, percentage of hemoglobin F, and platelet count at the time of diagnosis were 4.6x109/L (range, 0.2–31.6), 21% (range, 0–68), and 61.0x109/L (range, 1.4–483), respectively. The median observation period was 18 months (range, 1–287). During observation, 56 of the 92 patients received allogeneic hematopoietic stem cell transplantation (HSCT), and 30 of 92 patients died. Outcomes were assessed according to transplantation-free survival (TFS), in which HSCT and death were censored, and overall survival (OS) by the Kaplan-Meier method. Aberrant methylation of BMP4, CALCA, CDKN2A, CDKN2B, DMR-H19 and RARB were detected, of which hypermethylation of BMP4, CALCA, CDKN2A, and RARB were associated with poor TFS according to univariate analyses (P The present study shows that aberrant methylation of four genes (BMP4, CALCA, CDKN2A, and RARB) is associated with poor outcomes in JMML patients. Patients with SETBP1/JAK3 mutations frequently show the hypermethylation of these genes. Further, allogeneic HSCT is associated with improved outcomes for patients with AMS = 1 and 2. Therefore, these results suggest that examination of the methylation pattern of these four genes may help guide clinical decisions for the management of patients with JMML. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

Abstract 170 Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods: We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation. Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers' protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results: For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases. Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion: Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

Abstract 352 Background: Autologous stem cell transplantation (ASCT) for MM leads to complete responses in ∼20–40% of patients but rare cures. Patients with rapid recovery of T cells post ASCT may have improved outcomes suggesting possible immune mediated tumor control. We have shown that adoptive T-cell transfers after ASCT for MM using vaccine-primed and ex-vivo costimulated autologous T-cells in combination with pre- and post-transplant immunizations using a tumor antigen vaccine (+ GM-CSF and montanide) led to vaccine-directed T-cell responses in about 1/3 of patients and enhanced recovery of polyclonal T and B cell counts and function. We hypothesized that addition of Poly-ICLC (Hiltonol®) – a TLR-3 agonistic vaccine adjuvant could increase tumor antigen vaccine responses through better priming and boosting. Methods: We report interim results of a Phase II clinical trial (NCT01245673) evaluating safety and activity of autologous T cells primed in-vivo with a MAGE-A3 multipeptide vaccine (Orphan Drug Designation: GL-0817) mixed with GM-CSF and Poly-ICLC (Hiltonol®) +/− montanide. Inclusion criteria included measurable disease or high-risk cytogenetics. MAGE-A3 is expressed in ∼50% MM cases and more frequently in relapsed/extramedullary/proliferative disease. The MAGE-A3 vaccine has 2 HLA-A2-restricted class I peptides and 1 promiscuous class II peptide linked to an HIV-1-TAT membrane translocation sequence (Trojan peptide) to enhance peptide presentation. Vaccine-primed T cells were collected by leukapheresis, costimulated and expanded ex-vivo using anti-CD3/anti-CD28 mAb conjugated beads. T-cells were infused at day +2 after ASCT followed by booster immunizations at days 14, 42, 90, 120 and 150 post-transplant. Lenalidomide maintenance was started at day +100. Patients were evaluated for MM responses in accordance with IMWG criteria at days 60, 100, 180 post-transplant and Q3 months thereafter. T-cell and B-cell responses to the vaccine were evaluated by IFN-g or IL-2 cytokine production (all patients), dextramer binding to CD8 cells (HLA-A2 positive patients) and ELISA antibody assays at days 14, 60, 100 and 180 post-transplant. Results: 25 patients were transplanted on study. At a median followup of 6 months (range 2–18 months), 24 patients are surviving while 1 patient relapsed at about day +60 and died. Four additional patients have relapsed at 7,9,18 and 18 months post-transplant, yielding a 1-year Kaplan-Meier EFS of 77%. Of the 16 patients evaluable for response at day 100, 7/16 (44%) had CR/nCR using the study enrollment (post-induction) myeloma markers as a baseline while at day 180, 7/13 (53%) had CR/nCR. T-cell infusions were well-tolerated with no probable/definite grade 3 or higher toxicities. Vaccinations were associated with > 50 mm injection site reactions (redness, induration or both) after 1 or more immunizations in the majority of patients. Two patients developed large and prolonged inflammatory reactions which evolved into sterile abscesses. These resolved over 2–3 months with conservative management but as a result montanide was eliminated from the vaccine formulation for patients 11–25. Thereafter vaccine reactogenicity was decreased with no additional sterile abscesses. Of 16 patients tested for immune responses to date, 2 patients were unevaluable due to poor sample viability. Dextramer staining demonstrated MAGE-A3-specific CD8 T-cells in 4/4 (100%) of evaluable HLA-A2+ patients. Cytokine production in response to MAGE-A3 stimulation was seen in 11/14 (79%) patients; responses usually peaked at day 100 or day 180. Robust MAGE-A3 antibody responses were detected in 7/9 patients who received montanide in the vaccine formulation but in 0/7 patients who did not. Conclusions: Combination immunotherapy using a MAGE-A3 multipeptide tumor antigen vaccine plus vaccine-primed and costimulated autologous T-cells after ASCT for MM is well-tolerated and associated with encouraging early clinical responses. The addition of Poly-ICLC (Hiltonol®) to the vaccine formulation was associated with a high frequency of post-ASCT T-cell functional responses. The combination of Poly-ICLC +GM-CSF + Montanide led to robust injection site reactions that were occasionally severe and prolonged. Elimination of the montanide reduced injection site reactogenicity but may also have compromised the B-cell responses to the tumor antigen vaccine. Disclosures: Rapoport: Gliknik, Inc: Research Funding. Stadtmauer:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Vogl:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Research Funding; Otsuka: Consultancy; Acetylon: Research Funding. Strome:Gliknik, Inc: Equity Ownership, Patents & Royalties, Research Funding. Salazar:Oncovir, Inc: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract 266 Introduction: Lenalidomide is an immunomodulatory drug with effects on the innate immune system that may enhance antibody-dependent cell mediated cytotoxicity as well as the development of specific anti-tumor immune responses. These immunologic effects may synergize with the action of rituximab. To test the efficacy of lenalidomide combined with rituximab, we are conducting a single center, open label phase II clinical trial in patients (pts) with indolent B-cell or mantle cell lymphomas previously resistant to rituximab. Patients and Methods: Eligible pts must have relapsed/refractory indolent B-cell or mantle cell lymphoma with measurable disease that has failed to respond or has progressed within six months of a standard course of rituximab monotherapy (375 mg/m2 weekly for at least four weeks) or a prior rituximab-containing chemotherapy regimen. Thus, all pts enrolled are considered rituximab refractory. Patients were enrolled sequentially into two treatment cohorts. In Cohort 1, pts received two 28-day treatment cycles of lenalidomide 10 mg every day and dexamethasone 8 mg once weekly (Part I: lenalidomide + dexamethasone). After assessment of response to Part I, all pts received a single course of rituximab 375 mg/m2, consisting of four weekly doses during cycle 3 (Part II: lenalidomide + dexamethasone + rituximab). Treatment with lenalidomide + dexamethasone continued during and subsequent to rituximab; stable and responding pts continued on lenalidomide + dexamethasone until disease progression or development of clinically unacceptable toxicity. Response assessment after Part II was performed three months after the first dose of rituximab. In Cohort 2, dexamethasone was eliminated; otherwise, eligibility and treatment were the same. Results: As of August 9, 2011, 45 pts have started therapy (Cohort 1, n = 27; Cohort 2, n = 18); diagnoses include: follicular (n = 28), mantle cell (n = 11), small lymphocytic (n = 4), and marginal zone (n = 2) lymphomas; median age is 58 years (range: 35–85); male: female ratio is 31:14; median number of prior therapies is 3 (range: 1 – 7); LDH is increased in 18%. For Cohort 1, 24 pts completed Part II and are evaluable for response; 3 pts are not evaluable, 1 due to death (myocarditis) and 2 due to removal from study (1 thrombocytopenia attributed to myelodysplasia; 1 rash attributed to lenalidomide). For Cohort 2, 11 pts are evaluable for response assessment after Part II; 2 pts are not evaluable due to discontinuation of protocol therapy (1 rash attributed to lenalidomide; 1 with early symptomatic progression of lymphoma); 5 pts have not completed Part II. For 35 pts completing Parts I and II, at a median follow-up of 11.8 months, PFS is 73% (95%CI: 53 – 86%). Overall response rate (ORR) after Part I is 37% (6 CR; 7 PR); ORR after Part II is 60% (12 CR; 9 PR). Overall response rates after Part II do not differ between Cohort 1 (with dexamethasone) and Cohort 2 (without dexamethasone) [58% vs. 64%, respectively; p = 0.5]. There were fewer dose interruptions during Part I in Cohort 1 (15%) versus Cohort 2 (50%) [p = 0.02], reflecting reduced episodes of tumor flare and rash in Cohort 1. Conclusions: For rituximab-resistant pts with indolent B-cell or mantle cell lymphomas, the combination of continuous daily lenalidomide and a single four week course of rituximab, with or without low-dose weekly dexamethasone, achieves a high overall response rate with relatively durable responses. The addition of low-dose weekly dexamethasone may improve tolerance without decreasing the efficacy of the lenalidomide-rituximab combination. Disclosures: No relevant conflicts of interest to declare.

8060 Background: Lenalidomide may enhance anti-tumor immune responses by inhibiting Tregs and by augmenting NK-cell mediated antibody-dependent cytotoxicity. These immunologic effects may synergize...

Background Fludarabine-based therapies prolong survival of patients (pts) with chronic lymphocytic leukemia (CLL), but deplete T-cells causing immunosuppression which increases the risk of infection and may limit control of minimal residual disease by the immune system. Adoptive immunotherapy using autologous CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) enhances immune reconstitution after fludarabine-based therapy of lymphoma (Schuster Blood abstract 2007). Methods We conducted a multicenter phase I/II trial of ACTC following fludarabine- or alemtuzumab-based therapy of CLL. Eligibility required IWCLL 2008 indications for treatment of CLL. After leukapheresis, pts received fludarabine- or alemtuzumab-based therapy. Eight - 12 weeks after last immuno-chemotherapy, responding pts (CR, PR) received a single dose of ACTC prepared from autologous T-cells collected before therapy. Primary study endpoints were feasibility, defined as the ability to generate a T-cell dose of 1.0 E+10+/- 20%, and safety, defined as Results Thirty-four pts with CLL were enrolled (median age: 62). Eighteen patients were previously untreated and 16 patients had relapsed or refractory CLL. Four pts (12%) had del(17p), 11 pts (33%) del(11q), and 3 (9%) both del(17p) and del(11q). For previously untreated patients, 17 received fludarabine-cyclophosphamide-rituximab (FCR) and 1 pt received fludarabine-rituximab (FR); for relapsed patients, 10 received FCR, 3 FCR-bevacizumab, 2 alemtuzumab, and 1 oxaliplatin-fludarabine-cytarabine-rituximab (OFAR). Ten patients did not receive ACTC infusion for the following reasons: 6 pts (including 1 receiving alemtuzumab) had progressive disease prior to ACTC, 1 pt developed autoimmune hemolyic anemia during FCR, 1 pt developed ITP during FCR, 1 pt developed pure red cell aplasia during FCR, and 1 pt receiving alemtuzumab was removed from study per physician preference. Median ACTC dose was 1.0 E+10 CD3 cells (range: 3.94 E+08 -1.03 E+10). Two pts had progressive disease less than 90 days after ACTC and 1 pt was lost to follow-up. Twenty-one pts (14 CR; 2 CRi; and 5 PR at the time of ACTC infusion) were evaluable for studies of immune reconstitution at ≥90 days after ACTC. After chemotherapy prior to ACTC (D0), median CD4 count was 120 (range: 19-573); 90 days after ACTC (D+90), median CD4 count was 399 (range 62-1818) (p = 0.0003; median fold CD4 increase 2.3, range -0.5–20.9) [figure 1]. Median CD8 count also increased significantly (p = 0.004; median D0 = 86, range: 4-682; D+90 = 267, range: 18-2876; median fold CD8 increase 1.1, range: -0.8-18.4) [figure 2]. The increases in CD4 and CD8 counts between D0 and D+90 were not dependent on ACTC dose. There were no SAEs or grade 3 or higher non-hematologic toxicities related to T-cell infusion. For all 21 patients evaluable for immune reconstitution at D+90, median follow-up is 33 months with median PFS 30 months (not shown). PFS for previously untreated patients is 42 months, whereas PFS for relapsed patients is 12 months (figure 3). There was no correlation between magnitude of increase in post-infusion CD4 and CD8 counts and PFS at 1 year. Conclusions For CLL patients, ACTC production is feasible. ACTC infusion is well tolerated and results in a dose-independent acceleration of CD4+ and CD8+ cell recovery after fludarabine-based chemotherapy compared to historical controls. The application of adoptive immunotherapy with ACTC to accelerate immune reconstitution after immunochemotherapy could potentially provide an optimal immunologic milieu for early application of additional immunotherapeutic interventions. Disclosures: Levine: Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June: Novartis: Patents & Royalties, Research Funding.

Abstract 674 Immune recovery is an important determinant in multiple outcomes following allogeneic hematopoietic stem cell transplant (allo-HSCT). Delays in B and T cell reconstitution are associated with an increased risk of infection, relapse and secondary malignancy. Strategies to enhance post-transplant T-cell reconstitution could therefore improve morbidity and mortality after allo-HSCT. The cytokine Interleukin-7 (IL-7) is a unique therapeutic candidate to promote immune reconstitution because it has a central role in T cell development and survival. Murine models of allo-HSCT have demonstrated that IL-7 can enhance thymopoiesis as well as promote peripheral T cell survival and expansion. Initial clinical trials performed with recombinant human IL-7 (rhIL-7) have demonstrated a dose-dependent expansion of CD4+ and CD8+ T cells with an acceptable toxicity profile in patients with solid tumors or HIV infection. Hence we are conducting a phase I trial of post-transplant administration of rhIL-7 (CYT107, Cytheris Inc) in recipients of a T cell depleted (TCD) allo-HSCT to determine the safety, toxicity and biological activity on T cell reconstitution. To date, 9 patients (AML=7, MDS=2), with a median age of 59.3 years (range 27–67 years) have been treated with escalating doses of rhIL-7 (3 at 10 mcg/kg, 6 at 20 mcg/kg) administered subcutaneously weekly for 3 weeks following TCD allo-HSCT from an HLA compatible donor. Accrual is ongoing in the final cohort (30 mcg/kg). Recombinant hIL-7 was started at a median of 96 days post allo-HSCT (range 61–244 days). Most patients experienced transient minor injection site reactions. One patient (20 mcg/kg) developed a biopsy proven hypersensitivity drug rash a week after the first injection and was removed from the study (evaluable for toxicity but not immune recovery endpoints). No other significant injection-related toxicities have occurred, and no patients have developed GVHD. No anti-IL-7 antibodies or neutralizing antibodies have developed following rhIL-7 injection. Two of 9 patients with high-risk AML have relapsed (4 and 9 months post rhIL-7), an incidence consistent with published data in patients undergoing allo-HSCT for AML in CR, irrespective of T-cell depletion. Eight patients remain alive with a median follow-up of 14.5 months post rhIL-7 administration. At baseline, the median T cell counts were 91/mm3 (range 5 – 219 /mm3), 43/mm3 (range 9 – 299 /mm3) and 0 (range 0 – 17 /mm3) for CD4+, CD8+ and CD45RA+ T cells, respectively. Preliminary assessment of the immunological effects of rhIL-7 in 8 evaluable patients has demonstrated an increase in CD4+ T cells exhibiting a naïve or central memory phenotype (69% median increase over baseline at day 21 – range 8% to 35-fold increase), and CD8+ T cells exhibiting a naïve or effector memory phenotype (94% median increase over baseline at day 28 – range 0 to 11-fold increase). There was no observed effect on the frequency of CD4+CD25+FoxP3+ T cells or CD19+ B cells. TCR excision circles (TREC) analysis performed on CD4+ and CD8+ subsets in the first 6 patients, using absolute quantification real-time PCR, demonstrated increases in TRECs in 5/6 patients indicating enhanced T cell production. Finally, all 3 CMV-seropositive patients developed CD8+ T cell CMV-specific responses detected by intracellular IFNγ production to overlapping CMV-pp65 pentadecapeptides peptide pools after administration of rhIL-7. In one patient, we also analyzed CMV-specific T-cell frequency using HLA-A*0201 restricted MHC-tetramers. The highest CMV-specific response levels were noted in this patient with a history of CMV viremia and low-level CMV-specific CD8+ T cells prior to rhIL-7 (5.3-fold increase to the A0201-restricted immunodominant NLV peptide by tetramer assay after rhIL-7). Our pre-clinical data and early clinical results suggest that administration of rhIL-7 in recipients of a TCD allo-HSCT has minimal toxicity and can enhance post-transplant immune recovery without causing GVHD. Disclosures: Perales: Cytheris: Research Funding. Croughs:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morre:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van den Brink:Cytheris: Research Funding.

Abstract 420 Juvenile myelomonocytic leukemia (JMML) is a distinct subtype of myelodysplastic syndrome/myeloproliferative disorder (MDS/MPD) which, in analogy to chronic myelomonocytic leukemia (CMML), is characterized by excessive proliferation of myelomonocytic cells, but unlike CMML it occurs in young children and shows characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Chromosomal defect are present in 22% of patients in particular involving del7/7q. Mutations of genes involved in GM-CSF signal transduction, including RAS and PTPN11, can be identified in a majority of children with JMML, constitutional mutations of NF1 can be found in another 10% of patients with JMML, but in significant proportion of patients no molecular lesions were identified. To further clarify the molecular pathogenesis of JMML we have applied a high density SNP array-based karyptyping and screened for associated mutations including established defected in RAS, PTPN11 and NF1 but also c-Cbl and TET genes, recently identified in patients with CMML and MDS/MPD. We studied 49 children with JMML diagnosed between 1988 and 2008 in 28 institutions throughout Japan. The median age at diagnosis was 28 months (range, 1-75 months). Karyotypic abnormalities were detected in 11 patients, including 7 patients with monosomy 7. Two children had clinical evidence of NF1 mutations. Out of 49 patients, 32 received hematopoietic stem cell transplantation (HSCT). We performed mutational analysis of the genes known to be affected by mutations in JMML. PTPN11 mutations were found in 26/49 (53%) while NRAS and KRAS mutations were found in 2/49 (4%) and 1/49 (2%), respectively. None of the patients screened showed the presence of TET2 mutations, previously shown to be present in a significant proportion of patients with MDS/MPD, including CMML. High-density Affymetrix 250K single nucleotide polymorphism array (SNP-A) were applied as a karyotyping platform to identify LOH and submicroscopic copy number changes. Signal intensity was analyzed and SNP calls determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number and areas of UPD were investigated using Hidden Markov Model and CNAG v3.0 software. Compared to the results of conventional metaphase cytogenetics (MC), SNP-A identified significantly more genetic abnormalities (25% vs 49%; p=.02). In 1 patient UPD17q was present ivolving NF1 locus. In 4 patients UPD11q involving c-cbl locus (11q23.1) was found. Sequencing of c-Cbl gene family revealed mutations of c-Cbl in 5/49 (10%), and no Cbl-b mutations. All but 1 mutations were homozygous and were located in the RFD (exon 8 and intron 8). C-Cbl mutations were mutually exclusive with PTPN11, NRAS, and KRAS mutations or had clinical diagnosis of NF1. Unlike in CMML, no UPD4q24 or homo- or heterozygous TET2 mutations were found. Histomorphologic analysis did not reveal any distinct c-Cbl mutation-associated features or differences in count between patients grouped based on the presence of specific mutations. Similarly, there were no differences in gender, or the presence of cytogenetic abnormalities and the probability of 2 year overall survival of c-Cbl mutant cases between patients grouped according to mutational status. All patients with c-Cbl mutations displayed GM-CSF hypersensitivity at initial presentation but did not differ in this feature from the remaining JMML patients. However, mutant c-Cbl cases showed earlier presentation (median age 12 months vs. 29 months, p = .037) and lower median hemoglobin F fraction (3.5 % vs. 25%, p=.02). In sum, c-Cbl mutations constitute a novel important pathogenic lesion in JMML. While their presence suggests functional similarity to CMML, absence of TET2 mutation in JMML and rarity of PTPN11 mutations in CMML constitute important distinctive features of both diseases. Disclosures: No relevant conflicts of interest to declare.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by specific hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Mutations in RAS, NF1, or PTPN11 positioned in the GM-CSF signal transduction pathway, which contribute to myeloid proliferation, have been recognized in the pathogenesis of JMML. Recently a multi-step model for leukemogenesis has been proposed. In this model, the pathogenesis of leukemia requires at least two classes of mutations:primary mutations of genes implicated in cell differentiation such as AML1 and PU.1 andadditional mutations of genes contributing to myeloid proliferation such as FLT3, RAS, and PTPN11. We hypothesized that in patients with JMML, in addition to known mutations of genes in the GM-CSF pathway involved in myeloid proliferation, potential causative mutations of other classes might be acquired. AML1 encodes a transcription factor that is essential for definitive hematopoiesis, and its mutations have recently been found in adults with acute myeloid leukemia and myelodysplastic syndrome. However, no information is available on the profiles of mutations in these genes and the relationship between these mutations and clinical features of JMML in children. We analyzed mutations of N-RAS, K-RAS, and PTPN11 in 50, and of AML1 in 30 Japanese children with JMML by direct sequencing. Correlation between the mutational status and clinical and laboratory findings, including age at diagnosis, sex, fetal hemoglobin (HbF), platelets count, cytogenetic abnormalities, and hepatomegaly, all which are suggested prognostic factors for JMML, were also assessed. Of the 50 patients with JMML, cytogenetic abnormalities were detected in 14, including 8 with monosomy 7. PTPN11 and N-/K-RAS mutations were found in 24 (48%) and 7 (14%) patients, respectively, and a novel mutation in AML1 was identified in one patient. No simultaneous mutations in these genes were found. Age at diagnosis was older (median 36 vs 11 months, p=0.0005) and HbF level was higher (31.0% vs 5.1%, p=0.033) in patients with the PTPN11 mutation than those with the RAS mutation. No difference was observed between patients with PTPN11 and RAS mutations in sex ratio, white blood cell count, platelets count, and the incidence of cytogenetic abnormalities and hepatomegaly. Our results suggest that the clinical outcome of patients with PTPN11 might differ from those with RAS mutations because a higher level of HbF and older age have been reported to be poor prognostic factors. In one patient with JMML, we identified a novel mutation in the AML1 gene that belongs to the class of genes contributing to cell differentiation instead of the class of genes in the GM-CSF pathway involved in myeloid proliferation.

Juvenile myelomonocytic leukemia (JMML) is a rare, clonal myeloproliferative disorder afflicting young children and is characterized by specific hypersensitivity of JMML cells to GM-CSF in vitro. The pathogenesis of JMML seems to arise from dysregulation of GM-CSF signal transduction. Approximately, 75% of patients with JMML exhibit dysregulation due to mutations in RAS, NF1 or PTPN11, all of which are positioned in the GM-CSF pathway. Potential causative mutations in remaining patients are anticipated to be in signaling components downstream of the GM-CSF receptor. Recently, a somatic point mutation (JAK2 V617F) in the JAK2 gene, which plays a major role in the development of hematopoietic cells by transducing signals from growth factor receptors including GM-CSF receptor, was found commonly in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. All these myeloproliferative disorders are also characterized by hypersensitivity of hematopoietic progenitor cells to several growth factors and cytokines. Because the frequency of JAK2 V617F mutation in JMML remains unknown, we performed mutational analysis of JAK2 in 39 Japanese children (aged 1 to 69 months) with JMML to assess the pathogenetic relevance of this mutation in JMML, in addition to mutational analysis of PTPN11 and N-RAS. Genomic DNA were prepared from bone marrow or peripheral blood samples taken at initial diagnosis. Mutations of JAK2 (exon 12), PTPN11 (exon 3 and 13) and N-RAS (exon 1 and 2) were screened by direct sequencing. PTPN11 or N-RAS mutations were found in 19 (49%) or 3 (8%) patients respectively, and this is compatible with previous reports. In this study, no JAK2 V617F mutations were detected in any of the 39 JMML patients. In conclusion, we confirmed the presence of PTPN11 and N-RAS mutations in JMML. However, the JAK2 V617F mutation appears to be uncommon in patients with JMML, while it is extraordinarily common in classic MPD. Future investigation should focus on other signaling components in the same signaling pathway as JAK2, or on other parallel signal transduction cascades of the GM-CSF receptor.