FAB M2 Research Articles

Incidence of Germline Mutations in Cancer-Predisposition Genes in Children with Hematologic Malignancies: a Report from the Pediatric Cancer Genome Project

▪Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies.To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated.Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in >50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line.The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia.In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene. DisclosuresNo relevant conflicts of interest to declare.

CD25 Expression Is Associated with Inferior Clinical Outcomes in Elderly Patients with Acute Myeloid Leukemia

Background: Elderly patients with acute myeloid leukemia (AML) show unfavorable biologic characteristics and inferior clinical outcomes compared to younger patients. Because some elderly patients can benefit from intensive chemotherapy, the identification of prognostic markers is important for therapeutic decision-making. CD25 (interleukin 2 receptor alpha) has been reported to be highly expressed in leukemia stem cells and correlate with adverse outcomes in younger (less than 60 years) patients with AML. However, the significance of CD25 expression in elderly patients with AML has not yet been investigated.Methods: We retrospectively reviewed 154 newly diagnosed AML (de novo, secondary) patients aged 60 years or over (median: 69, range: 60-85 years), admitted between 2005 and 2013 to Jichi Medical University Hospital. Blast cells from bone marrow at diagnosis were identified by flow cytometry (FCM) using CD45/side scatter properties. CD25 expression in the blast region was analyzed by FCM combined with single- and two-color staining.Results: The two-color FCM analysis showed that CD25 was expressed in CD34, CD117, and CD13-positive blast cells. The median CD25 expression of the blast cells was 0.7% (range: 0-75.3%). On the basis of CD25 expression in bone marrow blasts from healthy volunteers (n=5; median, 3.2%; range, 1.5-5.6%), we defined CD25 expression >10% as positivity. In this setting, we identified CD25-positive blasts in 21 patients (14%). CD25+AML was characterized by the MDS/MPN history (30%) and, a low or zero frequency of favorable cytogenetics (0%), FAB M2 subtype (5%), and FAB M3 subtype (0%) compared with CD25-AML (n=133). Among the 118 patients who underwent induction chemotherapy, including 75 patients who received a combination of anthracyclin and cytarabine (Ara-C), 69 (58%) achieved complete remission (CR). CD25+AML was associated with an inferior CR rate (20 vs. 64%, respectively, P=0.003), inferior event-free survival (EFS) (1-year EFS, 0 vs. 33%, respectively, P< 0.001), and inferior overall survival (OS) (2-year OS, 13 vs. 38%, respectively, P=0.003) compared with CD25-AML. On multivariate analysis using the following factors: cytogenetics, age (over 75 years), MDS/MPN history, and intensity of induction chemotherapy, CD25-positivity was a significant risk factor for the CR rate (HR: 8.0, 95% CI: 1.7-36.6; P=0.008) and EFS (HR: 2.3, 95% CI: 1.1-4.7; P=0.021). For CD25+AML, the CR rate was higher with induction chemotherapy using a regimen of low-dose Ara-C combined with granulocyte colony-stimulating factor (50%) than that using a regimen of daunorubicine and Ara-C (0%). However, there was no significant difference in OS between patients with CD25+AML treated with induction chemotherapy and those who treated with best supportive care (2-year OS, 13 vs. 11%, respectively, P=0.29). There were no patients with CD25+AML received stem cell transplantation due to disease progression.Conclusion: This study demonstrated that CD25 is an independent prognostic factor for elderly AML patients. The prognosis of CD25+AML patients is extremely poor; therefore, it is necessary to develop alternative approaches, such as a regime including anti-CD25 antibody, therapy targeting leukemia stem cells, and nonmyeloablative stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Integrated Nuclear Proteomics and Transcriptomics Identifies New High Frequency Dysregulated Transcription Factors in AML

Acute myeloid leukemia (AML) is characterized by developmental arrest which is thought to arise from transcriptional dysregulation of myeloid development programs. Transcription factor (TF) dysregulation has been identified at both the genomic and transcriptomic levels; however the actual transcriptional environment in AML patients characterized by the relative abundance of TF protein expression compared with normal blasts has not yet been described. Here, we have analysed the nuclear proteome of AML blasts in comparison with normal CD34+ cells and carried out a parallel analysis of the transcriptome using Affymetrix arrays.In our experimental design we restricted our analysis to the minimally differentiated FAB M1 since this subtype has little developmental heterogeneity and would also be developmentally matched to normal controls. The final experimental design comprised nuclear protein extracts from 5 normal CD34+ controls and 15 FAB M1 patients (>80% viable blasts; <10% CD14+/CD15+ cells). Purity of nuclear fractions was assessed by western blotting for histone and GAPDH (Figure 1). Nuclear tryptic peptides were generated and in each experiment labelled with 8 channel isobaric tagging to allow relative quantification coupled with peptide/protein identification using tandem mass spectrometry. In parallel mRNA from these samples were analysed using Human Transcriptome Array 2.0 (Affymetrix, USA).In total 6164 proteins were found and quantified. After applying appropriate quality control criteria 437 proteins were found to be significantly dysregulated between normal CD34+ cells and AML blasts. To identify frequently dysregulated proteins we selected those that consistently changed in at least 5 AML patients (± >1.2 fold). This yielded 188 proteins of which 112 (60%) were nuclear proteins. We identified 7 upregulated transcription factors in leukaemic cells compared to normal CD34+ cells; 3 of which had been previously associated with AML (CEBPA, STAT6 and WT1). Comparative analysis of mRNA of these changes showed that these increases were also significantly observed at the transcriptional level (Table 1). The remainder (DRAP1, NFIC, HMGB1 and HMGB2) had not been previously reported in AML, and none of the changes were seen at the transcriptional level indicating increased expression arose at post-transcriptionally or were due to an increased level of nuclear localization. We also identified 7 down-regulated TF, one of which had been previously associated with AML (DAZAP1) with the remaining being newly described abnormalities (MYEF2, NFIX, FUBP1, TARDBP, ILF2, ILF3). Again most of these changes (4 of 6) were not seen at the transcriptional level. We also observed changes in 15 heterogeneous nuclear ribonucleoproteins (hnRNP) affecting mRNA processing (including: A0, A1, A2B1, A3, AB, C, D, DL, PF, H1, H3, K, L, R and UL2) and we are currently examining whether their expression correlates with increased alternative splicing that we have observed in these patients from analysis of the exon arrays.These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression that would not have been seen at the mRNA level. We are performing in silico analysis to determine whether dysregulation of these TF give rise to corresponding changes in known target genes. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early Occurrence of Therapy-Related Acute Myeloid Leukemia (t-AML) with Monocytic Differentiation with Trisomy 8 Cytogenetic Abnormality after Radioactive Iodine (RAI) Treatment for Papillary Thyroid Cancer

Background: Although most t-AML occurs after chemotherapy +/- radiation, exposure to radioactive substances can be implicated, as in nuclear accidents. Thyroid cancer is the most commonly endocrine malignancy. The relative risk of second primary malignancy in thyroid cancer patients is higher in those treated with RAI than those without, with a notable 2.5-fold increase risk of leukemia. The excess leukemia risk is greater in patients < 45 yrs compared with older patients. Recent reviews have attempted to characterize cytogenetic and molecular abnormalities from RAI related t-AML. We present the case of a young woman who developed AML with monocytic differentiation and Trisomy 8 cytogenetic abnormality after RAI treatment for thyroid papillary carcinoma.Case Presentation: A 29-year-old Chinese woman presented with worsening fatigue and menorrhagia over 3 months. Initial workup revealed pancytopenia (WBC 1.7, Hb 8.3, Hct 24, PLT 44, ANC 0.7). She had a history of papillary thyroid carcinoma, classic type, treated with total thyroidectomy followed by RAI ablation with 99 mCi of I-131 2 years prior. She had been without evidence of residual/recurrent disease since then. Her only home medication was Levothyroxine and had no other toxic exposure. Physical exam was unremarkable. Serology was negative for HIV, Hepatitis B or C. Peripheral smear showed decreased WBC count, few circulating blasts, atypical monocytes, and decreased platelets. Bone marrow aspiration and biopsy showed marked hypercellular marrow (100%) replaced by sheets of myeloid appearing-blasts, increased medium/large size CD68+ monoblasts with moderate basophilic vacuolated cytoplasm, as well as promonocytes comprising 90% of nucleated marrow cells. This was consistent with AML with monocytic differentiation. Flow Cytometry confirmed presence of HLA-DR, CD56, CD64, CD2, CD5, CD4, and CD11+ myeloblasts (27% of total cells) with increased CD56+ monocytes (23%). Cytogenetic analysis identified trisomy 8. Molecular testing was negative for FLT3 ITD and FLT3 TKD mutations by PCR, and negative for PML-RARA rearrangement by FISH.The patient was started on induction chemotherapy with cytarabine 100 mg/m2 daily as continuous infusion on days 1-7 + Idarubicin 12 mg/m2 daily for days 1-3 (7+3 regimen). Repeat bone marrow evaluation on day 14 revealed complete histologic and cytogenetic response. She received three cycles of consolidation with high dose cytarabine 3 g/m2. She is presently awaiting identification of a matched donor for allogeneic hematopoietic stem cell transplantation.Discussion: t-AML is a distinct subgroup of AML in 2008 WHO classification for their attributed worse outcome than their de novo counterparts with the same genetic abnormalities [except inv(16)(p13.1q22), t(16;16)(p13.1;q22) or t(15;17)(q22;q12)], suggestive of biologic differences. However, these findings are largely derived from patients who received cytotoxic treatment with topoisomerase II inhibitors, alkylating agents and/or RT. In contrast, little is known about outcome and prognosis in t-AML from RAI. We present a young woman with RAI related t-AML with monocytic differentiation and trisomy 8 after a 2-year latency who responded to standard induction and consolidation chemotherapy. To our knowledge the monocytic differentiation with Trisomy 8 was previously unreported.Retrospective studies and case series suggest that median latency of RAI related t-AML is ~ 7 yrs, in bimodal distribution peaking at 1-3 years and later at 8-10 years. 68% of t-AML from RAI have abnormal karyotype, with chromosomes 7, 5, and 8 most commonly involved, notably del 7q. FAB M2, M3, and M4 represented the vast majority of cases. Compared to patients with other t-AML, RAI-related AML tend to have significantly inferior survival. This suggests a unique mechanism of secondary primary malignancy, possibly via the Na(+)/I(-) symporter, a membrane glycoprotein that mediates active iodide uptake in thyroid and other tissues.Conclusion: t-AML from RAI is an under-recognized and under-reported form of AML, with unclear mechanism, wide-spectrum cytogenetic abnormalities and histology, undefined dose-risk ratio, and decreased survival. As the incidence of thyroid cancer increases, and utilization of RAI becomes more frequent, further investigation is needed to better characterize this unique entity, early detection and its optimal treatment. DisclosuresNo relevant conflicts of interest to declare.

Maintenance with Histamine and IL-2 Induces a Marked Expansion of Activated CD56bright NK Cells in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), residual leukemic (stem) cells that escape initial chemotherapy are considered a major source of relapse. Clinical trials have used IL-2 for AML patients with the aim to reduce relapse rates by eliminating residual leukemic cells through activation of NK and T cells. However, monotherapy with IL-2 has led to disappointing results. Nevertheless, recent clinical trials have shown that the co-administration of IL-2 and histamine dihydrochloride (HDC) provides maintenance of remission in AML. Histamine suppresses the formation of reactive oxygen species (ROS) thereby protecting NK and T cells from ROS-induced dysfunction and apoptosis. In addition, IL-2 is considered to maintain the anti-leukemic activity of NK cells. However, the direct effect of this treatment on NK cell numbers and anti-AML activity has not been studied in detail so far.In this study, we analyzed the immunophenotype and function of NK cells in a cohort of 7 AML patients (FAB M2, n=1; M4, n=2; M4-eo with inv 16, n=2; M5, n=2) treated with HDC plus IL-2. All patients had received induction chemotherapy with daunorubicin (45 mg/m² i.v. days 1-3), cytosine arabinoside, ARA-C (2 x 100 mg/m² i.v., days 1-7) and etoposide (100 mg/m² i.v., days 1-5) as well as at least 3 cycles of consolidation chemotherapy with high dose or intermediate dose ARA-C (Sperr et al, Clin Cancer Res 2004;10:3965-3971 and Krauth et al, J Immunol 2006;176:1759-1768). After having achieved a complete hematologic remission, patients were treated with HDC (0.5 mg) plus recombinant IL-2 (0.9 x 106 IU) twice daily s.c. for 21 days per cycle. Blood was drawn before and during treatment with HDC plus IL-2.We found that after one week of treatment with HDC plus IL-2, NK cell numbers increased in peripheral blood (from 101.8 ± 28.25 cells/µl before therapy to 208.2 ± 38.27 cells/µl after therapy, p<0.05). In the NK cell fraction, we observed an astonishing increment of CD56bright NK cells in all treated patients (from 7.2±0.97% or 17.6±5.8 cells/µl before therapy to 38.8±4.4% or 104±19.4 cells/µl after therapy, p<0.05; see Fig.1A/B). The cytotoxic activity of the CD56bright cells, as determined by NK cell degranulation and target cell lysis using the cell line K562, showed a significant increase in comparison to cells obtained before treatment (p<0.05). This was associated with an increased expression of KIR as well as the activation markers NKp44 (see Fig.1C), NKp46, and CD25 on NK cells. Furthermore, we observed a significant increase in expression of CD56 on NK cells after treatment with HDC plus IL-2 in our AML patients (2.5 ± 0.55 fold increase in the mean fluorescence intensity of CD56, p=0.02), whereas CD16 expression did not change significantly. In addition, treatment with HDC+IL-2 also induced an increased proportion of circulating CD4+CD25highCD127low/neg regulatory T cells (Treg). Finally, in vitro stimulation of NK cells with histamine plus IL-2 mimicked the effects observed in vivo. Interestingly, the in vitro treatment was also associated with an increased expression of CD56 without altered expression of CD16, suggesting that this effect could be a specific and reliable indicator of in vivo responses of NK cells to HDC plus IL-2 therapy.In conclusion, treatment with HDC plus IL-2 causes a striking increase in CD56bright NK cells. These specifically expanded NK cells exhibit an activated phenotype with enhanced potential to kill leukemic cells. We propose that the maintenance of remission in AML patients treated with HDC plus IL-2 might, at least in part, be the result of an improved anti-leukemic NK cell function. [Display omitted] DisclosuresSperr:MEDA Pharma GmbH & Co. KG: Speakers Bureau. Valent:MEDA Pharma GmbH & Co. KG: Speakers Bureau.

Constitutively High Expression of CD137L on Primary MM Cells Could be Inhibited By Chemotherapy but Re-Emerged after Desease Relapse

Objective: Multiple myeloma (MM) is a kind of malignant monoclonal plasma cell disorder. CD137L molecules are important member of TNFsuperfamily. Under physiological conditions, CD137L express on the surface of various active APCs and participate in keeping the balance of immune system. Under pathological conditions, CD137L could express on the surface of various tumor cells. For example, in hematologic malignancies, about 35% of AML with FAB type M4 or M5, and most CLL, B-cell lymphoma cells express CD137L. CD137L molecules play an important role in the maturation process of B cells. Active CD137L can promote proliferation, differentiation and immune response of B cells. But when B cells transform to plasma cells, the majority of B antigens will be lost, such as CD19, CD20, HLA-DR. CD137L are also lost in the process of transformation. Our previous studies showed that there is no expression of CD137L and CD137 molecular on normal plasma cells. But CD137L were highly expressed on MM cell lines U-266, KMS-11, My-5, LP-1 and 8266. CD137L signaling promoted U266 cell proliferation remarkably, which could be blocked by Bortizomib. CD137L signaling also pushed MM cells in the G1 phase to enter the S phase. To understand the expression of CD137L and CD137 on MM primary cells，We examined the bone marrow specimens of MM patients, then analyzed the relationship between the expression of CD137L and the clinical characteristics of patients.Methods: Flow cytometry was used to detect the CD137L and CD137 expression. Markers of normal plasma cell were CD45+CD38+CD138+CD19+CD56-, Markers of abnormal plasma cell were CD45-/CD45lowCD38++CD138+CD19-CD56+. bone marrow specimens from 127 cases of MM patients treated in the First Affiliated Hospital of Soochow University from 2012 to 2013 and the normal control from 10 volunteers were collected with the consent of patients and volunteers. 45 patients were newly diagnosed, 72 patients were checked after treatment, 10 patients were relapsed and refractory; 68 patients were males, 59 patients were females; The median age was 58(36-73); 3 pts were D-S staging I, 14 pts II, 59 pts III; 25 pts were ISS staging I, 65 pts II, 35 pts III.Results: Normal plasma cells didn’t express CD137L and CD137 molecular .CD137L molecular but not CD137 were expressed on primary MM cells in all 45 de novo patients. The median level was 46.7 (3.6–96.7)%, significantly higher than that on normal plasma cells (P <0.05). A retrospective analysis had been made to find no correlation between CD137L expression and patients’ age, gender, type, DS stage, ISS stage , LDH, creatinine, serum calcium, AKP, M protein and extramedullary plasmacytoma. The CD137L expression of MM cells from 12 de novo patients treated with PAD were decreased gradually with the increase of treatment courses and improvement of efficacy. The expression of CD137L on MM cells in 79 patients grouped in CR (n = 12), PR (n = 51), recurrent (n = 16) were 4.5 (0–18)%，10 (−056)% (P<0.05), 30.5 (5.3–95)% (P = 0.00) respectively.Conclusions: CD137L molecular without CD137 were constitutively highly expressed on MM cell lines and primary MM cells, but hardly on normal plasma cells. There was no relationship between the expression of CD137L with patients’ clinical and biological characteristics. But our data showed that constitutively high expression of CD137L molecular on primary MM cells could be inhibited by chemotherapy but re-emerged after desease relapse. DisclosuresNo relevant conflicts of interest to declare.

The RUNX1 Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion

Background: In AML four types of acquired alterations of the RUNX1 gene have been described: 1. translocations involving RUNX1 leading to fusion genes such as RUNX1-RUNX1T1, 2. molecular mutations, 3. amplifications of RUNX1, 4. Partial or complete deletions of the RUNX1 gene.Aim: To determine the frequency of different RUNX1 alterations and to characterize the spectrum of accompanying genetic abnormalities.Patients and Methods: We screened 726 de novo AML patients (pts) for RUNX1 deletions (del) and translocations using a dual color break-apart probe covering the 5′ and 3′ part of RUNX1 (MetaSystems, Altlussheim, Germany) and in addition evaluated RUNX1 mutations (mut) by Sanger or next-generation amplicon deep-sequencing. Median age was 67 yrs (range: 18 to 100 yrs). For all patients cytogenetics was available and categorized according to MRC criteria (Grimwade et al. Blood 2010). Partial deletions of RUNX1 as detected by FISH were confirmed by array CGH (Agilent Technologies, Santa Clara, CA).Results: In 89/726 pts (12.3%) abnormalities of the RUNX1 gene were detected by FISH: 10 pts (1.4%) showed a deletion encompassing the whole RUNX1 gene while additional 9 pts (1.2%) showed a partial loss of one RUNX1 copy. A gain of one RUNX1 copy was present in 45/726 (6.2%) pts. In 3 pts a gain of the 5′ part of RUNX1 was accompanied by a loss of the 3′ part while in 2 pts one copy of the 3′ part was gained accompanied by a loss of the 5′ part. A translocation affecting the RUNX1 gene was detected in 31 pts (4.3%). The partner gene was RUNX1T1 in 29 pts and located on 16q13 and 18p11 in one pt each. One pt with a RUNX1 translocation also showed a 5′ RUNX1 deletion. In 110/726 pts (15.2%) a RUNX1mut was detected. Of these, 16 pts showed two and 5 pts three mutations in RUNX1. Thus, in total 136 mutations were detected in 110 pts: 58 (42.6%) were frameshift, 42 (30.9%) missense, 21 (15.4%) nonsense, 9 (6.6%) splice-site and 6 (4.4%) in-frame insertions/deletions. The RUNX1mut was homozygous in 15 pts, these were predominantly missense mutations (9/15; 60%). Within the subset of pts with RUNX1mut 2 harbored an additional RUNX1del and 9 pts a gain of a RUNX1 copy, while no RUNX1 translocation was present. In AML FAB type M0 both RUNX1mut and RUNX1del showed the highest frequencies (33.3% and 14.8%). 48.4% and 45.2% of cases with RUNX1 translocations were FAB type M1 and M2. While RUNX1mut were most frequent in the cytogenetic intermediate risk group (19.1%; favorable: 2.2%, adverse: 9.7%), RUNX1del were most frequent in pts with adverse risk cytogenetics (9.7%; favorable: 1.1%, intermediate: 0.8%). A comparable distribution was observed for a gain of RUNX1 copies (adverse: 19.4%, favorable: 4.5%, intermediate: 2.6%). With respect to additional molecular mutations all types of RUNX1 alterations were mutually exclusive of NPM1mut. Further, the frequency of DNMT3Amut and CEBPAmut was significantly lower in pts with RUNX1 alterations as compared to those without (14.3% vs. 34.3%; p<0.0001 and 6.4% vs. 13.4%; p=0.012). However, some striking differences between the different types of RUNX1 alterations were detected: ASXL1mut were significantly more frequent in pts with RUNX1mut (36.7%) but rather infrequent in pts with RUNX1del, gain and translocation (12.5%, 6.1%, and 6.7%). A comparable association was noticed for SF3B1mut which were frequent in RUNX1mut pts (23.8%) and rather infrequent in pts with RUNX1del, gain and translocations (0%, 10.5%, and 0%). In contrast, pts with either RUNX1del or RUNX1 gains showed a significantly higher TP53mut frequency (66.7% and 35.3%) as compared to RUNX1mut or RUNX1 translocated pts (7.1% and 4.8%). In the total cohort median overall survival (OS) was 18.7 months and differed significantly between the different types of RUNX1 alterations: for RUNX1 translocations, mutations, gains and deletions it was 35.5, 14.1, 12.4 and 4.3 months.Conclusions: 1. The RUNX1 gene is altered in 26% of AML. 2. All types of RUNX1 alterations predominantly occur in AML M0, M1 and M2 and are rare in the remainder AML. 3. They are mutually exclusive of NPM1 mutations and show a negative association with DNMT3A mutations. 4. While RUNX1 mutations were most frequent in patients with intermediate risk cytogenetics, RUNX1 deletions and gains were most frequent in patients with adverse cytogenetics. 5. Outcome differs significantly and is best in patients with RUNX1 translocations and worst in cases with RUNX1 deletions. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Prevalence of Extramedullary AML Detected By 18-FDG/PET-CT: Results from the Prospective PET-AML Trial

Introduction: Acute myeloid leukemia (AML) may present with either concomitant or isolated extramedullary (EM) AML. Data on the prevalence of EM AML are based on retrospective and clinical analyses which rely on findings from physical examination only or on coincidental findings in standard imaging procedures and range from 2.5 to 9.1% (Bakst et al., Blood 2011;118(14):3785-3793). Previous studies identified EM AML as a prognostic factor in patients with AML. 18Fluorodesoxy-Glucose Positron Emission Tomography – Computed Tomography (18FDG/PET-CT) is able to detect highly metabolic tissue and has proven efficacy in imaging studies for various types of malignant diseases. In a pilot study in patients with histologically proven EM AML, we were able to demonstrate a sensitivity of 90% using 18FDG/PET-CT imaging and found additional EM sites in 60% of the patients (Stölzel et al., Haematologica 2011;96(10):1552-1556). The aim of this prospective, observational study was to determine the prevalence of EM AML in patients with newly diagnosed or relapsed AML using 18FDG/PET-CT (ClinicalTrials.gov Identifier: NCT01278069).Patients and Methods: A total of 94 patients with AML (newly diagnosed AML, n = 85 and relapsed AML, n = 9) underwent total body 18FDG/PET-CT scans at diagnosis after giving informed consent. Patients with EM diagnosed by 18FDG/PET-CT underwent a second 18FDG/PET-CT scan after induction chemotherapy. The median age of all patients was 61 years (range, 27 to 79 years). Patients were included only if a delay of ≤ 5 days of initiation of induction- or re-induction chemotherapy was clinically justifiable to perform the study. 18FDG/PET-CT scans were performed using a Siemens Sensation 16 as part of a biograph (Siemens, Knoxville, TN, USA) with i.v. application of 18FDG (range of activity 223 to 433 MBq) and 120 ml i.v. contrast media Ultravist 370 (Bayer Schering Pharma, Leverkusen, Germany). Adverse reactions due to the application of i.v. 18FDG and i.v. contrast media did not occur.Results: Total body 18FDG/PET-CT imaging detected highly metabolic manifestations suggestive for EM AML in 21 patients (22%). During follow-up after induction chemotherapy, 18FDG-avid EM was still observed in 50% of all PET-positive patients. In comparison with PET-negative patients, those with PET-positive EM AML had a higher bone marrow blast count, a higher prevalence of FAB M5 morphology, higher peripheral WBC, higher serum LDH, and less frequent FLT3-ITD mutations. Sites of EM AML were connective tissue (n=4), parenchymal tissues (n=8), and lymph nodes (n=15). In patients with clinically overt EM AML (n=8) additional EM manifestations were detected in 62% (n=5). In 13 patients, samples from EM sites were obtained for histology review – in 10 patients histology confirmed the occurrence of EM AML in these sites, indicating a specificity of 77%. Importantly, in the remaining patients in whom histology could not confirm EM AML, concomitant malignant tumors were found. Extrapolating these results on the entire cohort, the prevalence of EM AML in newly diagnosed and relapsed AML is 17%.Conclusions: Our study is the first to prospectively evaluate 18FDG/PET-CT imaging for the diagnosis of EM AML. According to our results, 18FDG/PET-CT is a useful and safe tool in order to detect EM AML with a high specificity. We found a prevalence of EM AML of 17%. This is two- to eightfold higher, than in previously reported clinical studies. Integration of 18FDG/PET-CT-based imaging procedures as adjunct for response assessment in these patients seems to be important. Further development of other PET-based imaging procedures and integration in the setting of relapse after allogeneic hematopoietic stem cell transplantation might be necessary and will contribute to a better understanding of the biology and prognostic role of EM AML and the development of targeted treatment strategies in patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Higher Expression of Suppressor of Cytokine Signaling1 (SOCS1) in the Bone Marrow Is an Unfavorable Prognostic Factor in Adult Patients with De Novo Acute Myeloid Leukemia

IntroductionSuppressor of cytokine signaling1 (SOCS1) protein, which encodes a member of the signal transducers and activators of transcription (STATs)-induced inhibitors, takes part in a negative regulation of cytokine signaling. The mechanism of SOCS1 in tumor carcinogenesis is complex and remains to be defined. Till now, there have been no studies concerning the prognostic implication of SOCS1 expression in acute myeloid leukemia (AML).Methods and MaterialsA total of 223 adult patients with newly diagnosed de novo AML who had enough cryopreserved cells for analysis at the National Taiwan University Hospital were enrolled consecutively. SOCS1 expression in bone marrow (BM) mononuclear cells was analyzed by quantitative real-time polymerase chain reaction. The results were correlated with FAB subtypes, clinical features, cytogenetics, other genetic alterations, and clinical outcome.ResultThe median value of SOCS1 expression was used as the cut-off value to divide patients into lower- and higher-expression groups. Higher SOCS1 expression was closely associated with older age (P=0.032) but inversely related to FAB M1 subtype and t(8;21)(q22;q22). There was no difference in other clinical parameters, including sex, hemoglobin level, white blood cell (WBC) counts, blast counts, and lactate dehydrogenase level between the two groups. Compared to patients with lower SOCS1 expression, those with higher expression had higher incidence of CD7 and CD34 expression on leukemic cells. To investigate the interactions of SOCS1 expression and other genetic alterations in the pathogenesis of AML, a complete mutational screening of 17 genes was performed. Higher SOCS1 expression was closely associated with NPM1 mutation and DNMT3A mutation (33% vs. 14.4%, P=0.002 and 20.9% vs. 10.8%, P=0.044, respectively), but negatively associated with CEBPA mutation (5.4% vs. 18.9%, P=0.002).Of the 154 AML patients receiving conventional intensive induction chemotherapy, 112 (72.7%) patients achieved complete remission (CR). The patients with higher SOCS1 expression had a lower probability of achieving CR than those with lower SOCS1 expression (62.9% vs. 81%, P=0.001). With a median follow-up time of 37 months (ranges, 0 to 160), patients with higher SOCS1 expression had poorer overall survival (OS) than those with lower SOCS1 expression (median 20 months vs. not reached, P=0.004). The same was also true among the patients with intermediate-risk cytogenetics and normal karyotype. In multivariate analysis, higher SOCS1 expression was an independent poor prognostic factor for OS in total cohort (relative risk, RR 1.947, 95% CI 1.081-3.508, P=0.026) irrespective of age, WBC, cytogenetics, NPM1/FLT3-ITD and CEBPA mutation. In the 77 cytogenetically-normal patients, higher SOCS1 expression was still an independent poor prognostic factor (RR 2.410, 95% CI 1.012-5.738, P=0.047).Interestingly, a scoring system incorporating SOCS1 expression and six other risk factors, including age, WBC, karyotype, FLT3/ITD, and mutations of NPM1 and CEBPA, into survival analysis was proved to be very useful to stratify AML patients into different risk groups (P =0.002).ConclusionAML patients with higher SOCS1 expression had distinct clinic-biologic features and poorer outcome. BM SOCS1 expression may serve as a new biomarker to risk stratify the patients. DisclosuresNo relevant conflicts of interest to declare.

An Analysis of CNS2 Patients with AML: Do They Require Additional Intrathecal Therapy? a Report from Children’s Oncology Group Protocols AAML0531 and 03P1 and St Jude Children’s Research Hospital Protocol AML02

Introduction: Pediatric patients with acute myeloid leukemia (AML) require central nervous system (CNS) directed chemotherapy to minimize CNS relapse. Patients with AML who have CNS disease at diagnosis, defined as >5 white blood cells (WBC) with positive blast in cytospin, require more intensive CNS directed therapy to clear CNS disease and prevent CNS relapse. Those with <5 WBC but with a positive cytospin are designated as CNS2 disease. Those without any blast (negative cytospin) are designated as CNS1. The need for extra CNS therapy for CNS2 patients varies among treatment protocols, and consensus has not been reached as to the best CNS directed therapy for these patients. We studied the impact of intensive vs. standard management of CNS2 patients by comparing patients treated on the two most recent Children’s Oncology Group (COG) trials to those treated on the St Jude Children’s Research Hospital AML02 trial, where COG trials managed those with CNS2 disease more intensively, similar to CNS3, and AML02 treated such patients similar to CNS1. Both trials used a similar MRC-based chemotherapy backbone. This backbone gave intrathecal (IT) cytarabine at the beginning of each cycle of therapy except Capizzi II therapy, and on the AML02 protocol most patients received IT triple therapy. On both backbones, patients with CNS3 disease, and those on COG trials with CNS2 disease, received twice weekly IT cytarabine until the CSF was clear of blasts plus 2 additional treatments with a minimum of 4 IT treatments given. Thus, this analysis is the first comparison of outcome of CNS2 patients examining the variable of additional CNS directed therapy.Methods: Disease characteristics and clinical outcome for children who were CNS2 treated with intensive or standard intrathecal chemotherapy were compared across COG and SJCRH studies and compared to those who were CNS1 or CNS3.Results: Among the 1361 eligible patients enrolled on AAML03P1 and AAML0531 with available data for CNS status, 247 had CNS2 status. Of the 200 eligible patients enrolled on AML02, 47 patients had CNS2 status. On all protocols, patients with CNS2 status had a significantly higher incidence of Inv(16) compared to CNS1 patients (p<0.001 for both protocols). As well, patients with CNS2 status had a higher incidence of M4 FAB classification compared to CNS1 patients (p<0.001 for COG protocols and p=0.002 for AML02). On both protocols, patients who were CNS2 had similar incidence of Inv(16) compared to patients who were CNS3. In contrast, on the COG protocols the incidence of M4 FAB was similar among CNS2 and CNS3 patients, while on the AML02 protocol the incidence of M4 FAB was significantly higher in CNS2 compared to CNS3.The event free survival (EFS) for patients treated on COG protocols showed significant differences according to CNS status, with CNS1 patients having superior EFS compared to CNS2 and CNS3 patients (p=0.005), while on AML02, the EFS for CNS2 patients was significantly higher than CNS3 patients (p=0.04) and higher, but not significantly, than CNS1 patients (p=0.06). The overall survival (OS) for CNS2 patients treated on COG protocols was not significantly different from that of CNS1 and CNS3 patients (p=0.32), while on AML02, the OS was significantly higher for CNS2 patients compared to CNS1 (p=0.05) and CNS3 patients (p=0.02)(Figures 1 and 2). However, for the AML02 protocol, the Cox analysis showed that CNS status was not independently associated with EFS, OS, or cumulative incidence of relapseOn the COG protocols there were 101 patients with relapse involving CNS, of which 34 were CNS1, 32 CNS2 and 34 CNS3 with a 5 year CNS refractory/relapse rate of 4%, 13% and 28% respectively (p<0.001). One patient with CNS relapse was unknown for CNS group. Of the 101 patients who relapsed involving CNS: 34% were CNS1, 32% were CNS2, and 34% were CNS3.Conclusions: Patients with AML who have <5 white cells with blasts present in the cerebrospinal fluid at diagnosis may not require extra IT therapy but may be able to be treated in the same fashion as patients with no blasts in the CSF at diagnosis. Additional analyses will assist in determining which, if any, CNS2 patients require extra IT therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3890: MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival

Abstract Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. The main cause for this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with a stem cell-like character. These so-called leukemic stem cells (LSC) are thought to be responsible for relapse. We hypothesized that success of novel anti-AML therapies relies on functional manipulation of genes including microRNAs, resulting in elimination of leukemic (stem) cells while sparing residual co-existing normal hematopoietic stem cells (HSC). We aimed at identification of microRNAs differentially expressed between HSC, LSC and the AML bulk obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment. To that end, we described immunophenotypic markers that can distinguish LSC from HSC. Moreover, we identified that HSC have higher aldehyde dehydrogenase activity than LSC (Schuurhuis et al. Plos One 2013). Comparing the microRNA expression profile of LSC with that of HSC showed that microRNA-551b (miR-551b) is highly expressed in residual HSC in the AML bone marrow. To determine whether miR-551b is a HSC specific microRNA we purified stem and progenitor cell subsets from normal bone marrow and showed that miR-551b is the highest expressed in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitors. To investigate whether the expression of miR-551b is of clinical importance in AML we determined its expression in AML bone marrow samples (n=154) and showed that high miR-551b is associated with lower complete remission (CR) rates after the first cycle of induction chemotherapy, shorter relapse free survival and a worse overall survival. In line with miR-551b being a stem cell miRNA, high expression in AML was associated with an undifferentiated morphology (FAB M0). To shed more light on the functional role of miR-551b in AML we correlated the expression of miR-551b with overall gene expression in a large panel of AML patients. Many of the genes that highly correlated with miR-551b like; MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC signatures. In conclusion, miR-551b is specifically expressed in normal stem and multipotent progenitor cells and high expression in AML is associated with poor prognosis. Currently, our research focuses on the function of miR-551b in AML. Citation Format: David C. de leeuw, Fedor Denkers, Peter Valk, Iris de Rink, Ron Kerkhoven, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Linda Smit. MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2014-3890

GATA2 mutations in patients with acute myeloid leukemia-paired samples analyses show that the mutation is unstable during disease evolution.

Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1% of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.

Retinoic Acid Therapy Resistance Progresses from Unilineage to Bilineage in HL-60 Leukemic Blasts

Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10–20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or “precommitment” phase) or subsequently (the late or “lineage-commitment” phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage maturation block), followed by bilineage maturation block and progressive signaling defects, notably the reduced expression of Vav1, Fgr, and c-Raf.

Ploidy and clinical characteristics of childhood acute myeloid leukemia: A NOPHO-AML study.

We report the first large series (n = 596) of pediatric acute myeloid leukemia (AML) focusing on modal numbers (MN) from the population-based NOPHO-AML trials. Abnormal karyotypes were present in 452 cases (76%) and numerical aberrations were present in 40% (n = 237) of all pediatric AML. Among patients with an abnormal karyotype, the MN 46 was most common (n = 251; 56%) of which 36 (8%) were pseudodiploid with numerical aberrations, followed by MN 47 (n = 80; 18%) and MN 43-45 (n = 48; 8%). No cases had MN less than 43. Hyperdiploid AML with MN 48-65 comprised 11% of all cases and was associated with early onset (median age 2 years), female sex (57%), and a dominance of acute megakaryoblastic leukemia (AMKL) (29%). Hypodiploidy constituted 8% of all AML and was associated with older age (median age 9 years), male predominance (60%), FAB M2 (56%), and t(8;21)(q22;q22) (56%) with loss of sex chromosomes. Inferior outcome was observed for hypodiploid cases (5-year event-free survival 40% and 5-year overall survival 40%) but did not reach statistical significance. Chromosomes were gained in a nonrandom pattern, where chromosomes 8, 21, 19, and 6 were the most commonly gained. In conclusion, based on MNs, two cytogenetic subgroups with characteristic clinical features are described; hypodiploidy found in 8% and associated with high median age, male sex, t(8;21)(q22;q22), and FAB M2 and possibly associated with inferior outcome (P = 0.13), and hyperdiploidy with MN 48-65 in 11% associated with early onset, female sex, and AMKL.

DNMT3A R882 mutations in patients with cytogenetically normal acute myeloid leukemia and myelodysplastic syndrome

Several molecular markers have been described that help to classify patients with acute myeloid leukemia (AML), a heterogeneous hematopoietic tissue neoplasm, into risk groups. We determined the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, in primary, cytogenetically-normal AML (CN-AML) and CN-myelodysplastic syndrome (MDS). A total of 63 CN-AML and 16 CN-MDS patients were analyzed for mutations in DNMT3A, codon R822 by direct sequencing and mutation of NPM1 and FLT3/ITD. DNMT3A mutations were found in 17/63 (27%) of CN-AML and in 1/16 (6.3%) of CN-MDS patients. Patients with DNMT3A mutations were older (p=0.047), had higher white blood cell (WBC) counts (p=0.046), more often belonged to FAB groups M4 and M5 (p=0.017), and were more associated with NPM1 mutations (p=0.017), than those with wild-type DNMT3A. DNMT3A-mutated patients had shorter overall disease survival (p<0.001) and disease-free survival (p=0.014) when the entire patient cohort was considered, which remained significant in multivariate analysis. We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in CN-AML, less frequent in CN-MDS, and that testing for R882 mutations may provide a useful tool for refining risk classification of CN-AML.

Celiac Disease Transmitted By Unrelated Cord Blood Stem Cell Transplantation (CBST)

Celiac disease is due to intolerance to certain cereal proteins leading to immune-mediated small bowel villous atrophy and malabsorption. Specifically, the gliadin component of wheat, and the prolamin component of rye and barley are implicated in causing disease. BMT and CBST have been known to transmit immune associated diseases such as diabetes mellitus immune thrombocytopenic purpura from an affected donor to transplant recipient. We observed the occurrence of Celiac disease in a patient a year following cord blood stem cell transplantation (CBST) for acute myelogenous leukemia (AML- FAB M2) in complete second remission (CR-2). The patient had no history of celiac disease prior to CBST nor any family member. The cord donor was HLA-identical unrelated male donor with HLA types: A3, B7 (w6), DR (B1), DR (B5). The family history of the donor was unavailable for celiac disease. The CBST was complicated by grade 2 skin Graft versus Host Disease (GVHD), which responded to steroid therapy. A year post transplantation she developed persistent mucous diarrhea with tinge of blood associated with abdominal cramps. Investigations for infectious causes such as CMV enteritis were negative and colonsocopy did not reveal any evidence of GVHD. Gastrointestinal symptoms persisted and did not respond to steroid and prograf therapy. Subsequent duodenal and jejunal biopsy revealed subtotal villous atrophy with cryptic hyperplasia suggestive of celiac disease. Antigliadin IgA and IgG, reticulin IgA and Endomysial IgA andibodies were elevated. A diagnosis of post-CBST coeliac disease was made. She responded well to gluten-free diet and became symptom-free. A literature review identifies only from donor to recipient one previous example of transmission of celiac disease following HSCT. This case could be the second case of celiac disease following transplantation and the first one post CBST. An association of celiac disease with some HLA types, including DQA1.0501, and DQB1.0201, in conjunction with the haplotypes A30, B18, DR3, DRw52, and DQ2 was recently noted. The donor of this case exhibit HLA DQ A1.0501 and DQ B1.0201 alleles. The findings suggest transfer of celiac disease by cord stem cells and confirm the immune nature of the disease. In addition the propensity to develop T-cell non-Hodgkin lymphoma and transmission of celiac disease by CBST support T cell concept in celiac disease. Autoimmune enteropathy should be considered in patient with persistent diarrhea post hemopoietic stem cell transplant.

A Case of Acute Myeloid Leukemia (FAB M2) with Inversion 16 Who Presented with Pelvic Myeloid Sarcoma.

Acute leukemias are the most common childhood cancer in all age groups. Acute myeloid leukemias (AML) constitute about 15–20% of acute leukemias. Fatigability, pallor, fever, and bleeding are the most common presenting symptoms of AML. Hepatosplenomegaly and lymphadenopathy are commonly encountered during physical examination. In rare instances eruptions due to skin involvement and localized tumor masses (myeloid sarcoma) may be found. Myeloid sarcoma is especially seen in AML-M2 subtype. By cytogenetic analysis, in AML-M2 subtype t(8;21) is often seen and it is more probable to find inversion 16 in AML-M4Eos subtype. Herein, we present a 15-year-old girl whose initial symptom was abdominal pain for three days and her pathological sign was a large abdominal mass which was verified by imaging studies and diagnosed as myeloid sarcoma by biopsy. On bone marrow examination, she had diagnosis of AML-M2 and by cytogenetic analysis inversion 16 was positive. She was treated with AML-BFM 2004 protocol and she is being followed up in remission on her ninth month of the maintenance therapy.

BCOR and BCORL1 Mutations In Pediatric Acute Myeloid Leukemia

IntroductionIn pediatric acute myeloid leukemia (AML) current survival rates are approximately 70%, but further improvements are required to improve disease outcome. Prognosis is correlated to early response to treatment and genetic aberrations (Creutzig et al, 2012). In approximately 20% no cytogenetic aberrations can be identified. In some of these cases repetitive aberrations, such as NPM1 mutations or cryptic translocations including NUP98-translocations (Hollink, 2011 and De Rooij, 2013) have been found. Recently, mutations in BCOR and BCORL1, both located on the X-chromosome, were found in adult AML using next generation sequencing. They both are transcriptional co-repressors, although with distinct binding targets (Tiacci, Heamatologica, 2012), and are thought to represent a novel mechanism of leukemogenesis. Somatic inactivating BCOR mutations were identified in 4% of adult cytogenetically normal (CN-) AML patients, predominantly located in exon 4, but also in other exons (Grossmann et al, 2011). Of interest, germline BCOR mutations cause the X-linked oculo-facio-cardio-dental genetic syndrome, which may occur due to its function as a co-repressor of the BCL6 gene. Somatic inactivating BCORL1mutations were found in 6% of adult AML patients (Li et al, 2011); all mutations were located in exon 4. Their exact role in AML and the targets of their co-repressive transcriptional activity has not been elucidated as yet (Tiacci, Haematologica, 2012). MethodsWe screened newly diagnosed pediatric AML patients for the presence of BCOR and BCORL1 mutations using direct sequencing of the complete coding sequence of both genes starting with a cohort of 86 patients including all cytogenetic subgroups patients, and later expanding this with an additional 146 patients for BCORL1screening of exon 4. This cohort was enriched for samples from CN-AML patients (56% and 21% respectively). Samples were obtained from the Dutch Childhood Oncology Group (DCOG; The Hague, The Netherlands), the AML-BFM-SG; Hannover, Germany and Prague, Czech Republic, and the Hôpital Robert Debré (Paris, France). ResultsA single BCOR mutation was found in 1 patient only with CN-AML. The mutation, p.A854T, was located in exon 4. The patient was a 4 year old boy with a FAB M1, WBC 354 x 109/L, who is alive 45 months after diagnosis. In addition, only 1 patient carried a BCORL1 mutation. The mutation, located in exon 4, p.G158X, caused a premature stop-codon. The male patient was diagnosed with secondary AML, aged 17 years, with normal cytogenetics and a WBC of 9.4 x 109/L, FAB M1, and died 3 months after diagnosis. Multiple recurrent SNPs were observed for both BCOR (rs5917933: 7/86 pts (91.9%); rs6520618: 15/86 (17.4%); rs144606152: 6/86 (7.0%)) and BCORL1 (rs4830173: 232/232 (100%); rs5932715: 36/232 (15.5%)), all in exon 4. No relation could be found between the presence of SNPs and disease outcome. ConclusionsBCOR and BCORL1 mutations occur in less than 1% of pediatric AML patients. These data provide further evidence for the differences in genetic background between pediatric and adult AML. Separate next-generation studies should be performed to elucidate the genetic background of pediatric CN-AML.This project was funded by KIKA, project number 64, entitled: Aberrant signal transduction profiling in pediatric AML. Disclosures:No relevant conflicts of interest to declare.

Is Superinstability Of Karyotype Of Acute Myeloid Leukemia “Relic Radiation From Big Bang” Of Chromosome Homeostasis On MDS Stage?

IntroductionIn some cases of the cytogenetic analysis patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) there are increase frequency non-clonal disorders (“cytogenetic noise”) in combination with complex (≥ 3 chromosomal aberration) clonal disorders. It is not infrequently manifested in the form of superinstability of the karyotype. In this case every aberrant cell is genetic different from each other. It is obvious that super unstable karyotype has a poor prognosis even more than complex clonal disorders, since due to the high genetic instability is a strong possibility of mutations that cause resistance to chemotherapy. In other words, the high level of “cytogenetic noise” on background of complex clonal disorders can be used as a marker of highly aggressive course of AML and MDS. The aim of our study was to determine the effect of superinstability of the karyotype on the current and prognosis of AML and MDS. MethodsThus, 157 patients with cytogenetic survey from register of AML and MDS by Kirov Research Institute of Heamatology were included in our research. They are 73 men, 84 women have the age from 21 to 81 (Me 55) years. Among them 34 patients have MDS and 123 – AML excluding promyelocytic leukemia. Chromosomal aberration were determined from bone marrow aspirate by standard cytogenetic method (GTG-method). Was evaluated at least 20 metaphase cells. ResultsAmong MDS patients the prevalence of superinstability of the karyotype was 29%, while among AML – only 4%, χ2=16.9, p<0.0001. In MDS group 2 patients (20%) are RAEB-1, 7 (70%) – RAEB-2, 1 (10%) – unspecified MDS. In AML group 2 patients (40%) are M2 FAB, 1 (20%) – M4 FAB, 2 (40%) - unspecified AML. Given that this type of cytogenetic of high instability is a characteristic feature of MDS, we can suppose, that AML with such instability always have the preliminary MDS stage with cell superinstability of the karyotype. In other words, the superinstability of the karyotype in AML can be regarded as “relic radiation from Big Bang” of chromosome homeostasis, which occurred at the preliminary MDS stage. The median of overall survival among all these patients treated by standard chemotherapy was 3 month, maximum survival – 16 month. The median of overall survival in AML patients was 1 month, MDS – 3.5 months. All patients were died. ConclusionA chromosomal superinstability of tumor cells that greatly enhances the aggressiveness of the current AML and MDS, leading to chemotherapy resistance and dramatically reduces the overall survival patients. Superinstability of the karyotype determines the group of patients untreatable by standard chemotherapy. The allogeneic bone marrow transplantation need in these cases. Disclosures:No relevant conflicts of interest to declare.

Disease Characteristics and Prognostic Implications Of Cell Surface FLT3 Receptor (CD135) Expression In Pediatric Acute Myeloid Leukemia – A Report From Children's Oncology Group

FLT3 is a highly expressed cell surface receptor in a majority of acute leukemias with near universal expression in acute myeloid leukemia (AML). High FLT3 expression level in conjunction with 11q23/MLL-rearrangement has been associated with poor prognosis in pre-B acute lymphoblastic leukemia (ALL) and is considered a potential therapeutic target through FLT3 inhibition. In vitro studies have demonstrated enhanced sensitivity of ALL and AML cells with high FLT3 expression to the cytotoxic effects of FLT3 inhibition. Although the clinical impact of high allelic ratio FLT3/ITD has been demonstrated in AML, the significance of FLT3 expression in those without the mutation has not been well studied, and all previous studies have been limited to evaluation of FLT3 transcript levels for correlation with outcome. Here we present the results of a prospective evaluation of FLT3 cell surface protein expression (CD135) on the diagnostic leukemic blast population as determined by multi-dimensional flow cytometry (MDF) in specimens from patients treated on COG AAML0531. Of the 495 patients enrolled after September 25, 2008, 366 patients enrolled on the accompanying biology study and had diagnostic specimens for evaluation of central CD135 expression by MDF. All diagnostic specimens underwent central MDF analysis for CD135 expression. FLT3 mutation data was available on all 366 patients, 57 patients were FLT3/ITD (15.6%), 21 were FLT3/ALM (5.7%) and the remaining 290 were FLT3wild type (FLT3/WT).There was significant variability of CD135 expression across the population with a mean fluorescence intensity (MFI) ranging from 3-232 (median 18). Median CD135 expression for those with FLT3/ITD, FLT3/ALM, FLT3/WT was 22 vs. 19 vs. 17 respectively (p=NS). Patients were divided into quartiles based on CD135 expression and clinical characteristics and outcome were correlated with CD135 expression across the four quartiles. There was not a significant difference in CD135 expression by age, gender, race, or ethnicity across the four quartiles. Those with high CD135 expression (Q4) had similar median diagnostic WBC counts as those with lower CD135 (Q1-3), although median diagnostic blast % in those in Q4 was significantly elevated (p=0.003). Nearly half of those in Q4 were FAB M5 compared to 13% in Q1-3 (p<0.001) and all those with FAB M7 had low CD135 expression (p=0.004).Evaluation of the diagnostic cytogenetics and CD135 expression demonstrated significant correlation of CD135 expression with MLL translocations, as 22% of patients in Q4 had MLLtranslocations vs. 11% in Q1-3 (p=0.005). This was primarily driven by patients with t(9;11), p=0.001. There was an inverse correlation between inv(16) and CD135 expression as only 2% of those in Q4 had inv(16) compared to 15% in Q1-3 (p<0.001). CD135 was then correlated with response to induction and post-induction outcome. Complete remission (CR) rate was comparable between those with high or lower CD135 expression (p=0.76). Those with high or lower CD135 expression had similar 3-yr overall survival (OS) from diagnosis of 70% and 66% respectively (p=0.9) and relapse risk from CR of 42% and 36% respectively (p=0.35).Given the known prognostic and therapeutic significance of FLT3/ITD, we evaluated any prognostic implications of FLT3 expression in patients without FLT3/ITD, most of whom lack other cytogenetic risk features. Similar to the entire cohort, in the FLT3/ITD-negative cohort CD135 expression correlated with FAB M5 (p<0.001), t(9;11), (p<0.001) and inversely correlated with inv(16), p=0.001. 3-yr OS for those in Q4 was 71% vs. 67% in Q1-3 (p=0.955). Given the association of MLL and CD135 expression, we evaluated the association of CD135 expression with outcome in patients with MLLtranslocations (N=73); in these patients the 3-yr event free survival (EFS) was similar between those with high or lower CD135 expression (p=0.621).In summary, we found that CD135 surface expression did not correlate with FLT3 mutations or clinical outcomes. Although FLT3 expression in FLT3/ITD-negative patients does not appear to offer additional prognostic information with current therapy, it may provide a therapeutic target in a subset of high expressing patients. This study also demonstrates that elevated FLT3 expression is associated with MLL rearrangements and warrants further study in this population with regards to potential prognostic and therapeutic implications. Disclosures:No relevant conflicts of interest to declare.