ALBERT

Description: Background Acute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia (AML). However, a subset of APL patients is not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Some mechanisms such as increased ATRA metabolism have been suggested to acquired resistance to ATRA. However genetic mechanism of ATRA resistance has not been characterized at all. Hence, in this study, we tried to reveal genetic alterations attributable to ATRA resistance. Methods First, we performed whole exome sequencing (WES) using DNA of three APL patients who showed resistance to ATRA based treatment. These included two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite of consolidation treatment. DNA extracted from bone marrow at the time of diagnosis was used for analysis, while DNA extracted from saliva at the time of CR was used as germline control. Calling of single nucleotide variants (SNV) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNV’s found by WES were validated by direct Sanger sequencing. The frequency of those validated SNV’s was defined in a separate APL cohort. Results We identified 34 somatic non-synonymous SNV’s in three patients. Polyphen-2 algorithm predicted 9 among 34 SNV’s to damage protein function. Sanger sequencing revealed 8 over 9 SNV’s to be validated. These validated SNV’s include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, TNPO3 L173W, BHMT M185I and ADAMTS5 G85D. When these 8 SNV’s were genotyped in a separate cohort, none of these SNV’s was found in the APL cohort composed of 30 ATRA sensitive patients, suggesting these SNV’s would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics was performed, we observed 1) Increase in hydrogen bonding, 2) Decreased helix folding structure, 3) Decreased energy state in RXRG mutant case. Conclusions WES identified eight SNV’s which were unique in ATRA resistant cases. Among those mutations, RXRG could be a promising nonsynonymous mutation that explains the genetic mechanism of ATRA resistance. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4902 Introduction Cytogenetics and fluorescent-in situ hybridization (FISH) are important outcome predictors in multiple myeloma (MM). There were only few small studies that investigated prognostic implication of FISH and/or conventional karyotyping in Korean MM patients. We investigated the incidences and prognostic significances of chromosomal abnormalities detected by FISH and/or conventional karyotyping among Korean MM patients. Patients and Methods We collected data of patients from Korean Myeloma Registry and performed retrospective analysis. We compared the survival of patients with chromosomal abnormalities and other clinical findings. Results From 2000 to 2009, total of 801 newly diagnosed myeloma patients were enrolled in this study. Median age of patients was 62 years. Median overall survival was 82 months, and median follow up of time was 92 months. Among the patients who had conventional karyotype analysis, 17.1% were complex karyotype, followed by del13q (7.4%), hyperdiploidy (7.6%), hypodiploidy (3.0%), and t(11;14) (3.9%). Among the patients who had FISH analysis, 22.8% were del 13q, followed by t(11;14) (18.2%), t(4;14) (13.7%), del17p (11.8%) and t(14;16) (5.9%). Univariate analyses revealed that complex karyotype (p

Description: Purpose: von Willebrand disease (vWD) is the most common hereditary bleeding disorder and type 2B combines thrombocytopenia. So it must be considered in patients found to have low platelet counts, particularly if there is a family history of mucocutaneous hemorrhage. We performed this study to diagnose the type 2B vWD in chronic immune thrombocytopenic purpura (ITP) children. Methods: Seventeen cases among chronic ITP children over 6 months at the Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea from October, 1995 to June, 2007 were participated in this study. We performed screening coagulation tests such as platelet counts, activated partial thromboplastin time (aPTT), and bleeding time (BT) and specific tests for vWD such as von Willebrand factor related antigen (vWF: Ag), vWF ristocetin cofactor (vWF: RCo), factor VIII, and vWF multimer on these patients. These tests were also performed their family when the patient was diagnosed vWD. And we reviewed their past and family histories about bleeding tendency. Results: There were four boys and thirteen girls and their mean age was 11.6 years (range: 2.8∼18.5 years). Five cases (5/17, 29.4%) were diagnosed vWD: one had lower level of vWF: RCo and factor VIII with normal level of vWF: Ag and others had lower level of vWF: RCo and vWF: Ag with normal level of factor VIII. Among these, two cases showed abnormal screening test results, prolongation of aPTT or BT. We could perform the vWF multimer test in two cases, but two had normal pattern. Among five vWD children, we could obtain the past and family bleeding tendency histories except one case and three families showed bleeding tendency. But all families showed normal screening and specific test results. Conclusions: von Willebrand disease was combined in 5 cases (29.4%) among 17 chronic thrombocytopenic children. More evaluation such as vWF multimer and ristocetin induced platelet aggregation test (RIPA) is needed to confirm the subtype. And we should repeat the evaluation to the family who had bleeding history but showed normal results for diagnosis or exclusion of vWD.

Description: Background Familial form of acute myeloid leukemia (AML) is not well known except for extreme cases. BRCA1/2 and p53 germline mutation are well known genetic changes that is related to familial AML¡¯s. Because of diverse penetrance potential of germline mutations and complex pathogenesis for the development of AML, it is not easy to discern familial form of AML. In this study, we performed next generation sequencing (NGS) study of a family who has been suspected to have familial form of AML. Material and method Two relatively young patients have been referred to our institution for AML. One was a 41 years old male (son) and the other was 58 years old female (mother). To discover genetic factors involved in hematological familial cancer syndrome, we performed WGS/WTS using DNA of mother and son who are AML patients and WGS using DNA of healthy father. Tumor DNA and RNA of mother and son were extracted from bone marrow samples which were collected at the time of diagnosis and control DNA and RNA was extracted from saliva and bone marrow samples, respectively, at the time of CR. WGS were performed using HiSeq X ten (illumina, San Diego, USA). For WTS, we used HiSeq2000 platform (illumina, San Diego, USA). For the analysis of WGS, we used GATK unifiedGenotyper for caller in this study. The first filter was set an average depth 〉10 and conf_cut 〉 50. And next filter step was using several databases (dbSNP, clinvar, cosmnic70, nci60) and filtering tools (Meta SVM and Meta LR) to filter out of called loci. Meerkat was used to analyze structure variations (SVs). To analyze DEGs using WTS, we used HTSeq-count. To analyze DEGs, we used WTS and HTSeq-count. Results and discussion A total of 3,695,266 loci of normal DNA and 3,977,321 loci of tumor DNA were found in mom samples and 3,622,083 of these were commonly found in both normal and tumor samples. Similarly, a total of 3,513,806 loci of normal and 3,935,873 loci of tumor DNA were found in son samples, of which 3,476,405 were commonly found. Of these common loci, we identified 2,799,429 that are universally present in both mum and son. To determine genes that are found only in patients (Mon and son), we filtered out the loci of father¡¯s SNPs and excluded 2,191,882 loci We subsequently identified a total of 607,547 candidate loci which have association with AML malignancies. Using Support vector machines (SVMs) and DNM filter, we found 37 significant genes that are considered to be related with de-novo AML Among these, 12 were (AGL, COL12A1, IMPDH1, LIPN, MET, MYH13, PBX3, ROBO3, SLC34A3, SMO, THBS1 and TP63) already reported to have an association with hematopoietic disorder, while 25 were a novel mutation genes. The most interesting gene is THBS1. It is reported to affect hematopoietic differentiation function via CD36 and CD47 and a greater than 3 fold change was found in both mum and son in DEG analysis. In DEG analysis, total 1317 gene in mother and 473 genes in son were shown differently expressed above 3FC. The number of recurrent DEGs between mother and son is 144 and these genes were estimated to involve in Systemic lupus erythematosus, Chemokine signaling and bladder cancer pathway. We used Mutect to detect somatic mutation that acts as second hit. 29 nonsynonymous-SNVs were detected in son sample and 43 nonsynonymous-SNVs were detected in mother sample. OR11H1 gene was recurrently shown in both mother and son. We performed Structure variants analysis to identify second hit SVs using Meerkat in mother and son separately SVs of mother were detected in several regions and 69 genes were detected in exonic regions, 39 genes were somatic SVs and 12 of these were filtered out because the genes were also detected in the normal sample of son. 27 SVs genes are considered as a candidate of AML second hit in mother. On the other hand, we found 5 genes somatic SVs in son using the same method. Of note, FAM231A gene overlapped with mom tumor sample and son tumor sample. In conclusion, we found 37 significant genes may be related with de-novo AML. In addition, several genetic factors affect tumorigenesis through second hit. Figure 1. Association between THBS1 and other genes by gene to gene networking Figure 1. Association between THBS1 and other genes by gene to gene networking Figure 2. Expression level and heatmap of gene expression of mother and son by DEGs analysis Figure 2. Expression level and heatmap of gene expression of mother and son by DEGs analysis Figure 3. Circosplot of Structure Variantions(SVs) of mother and son Figure 3. Circosplot of Structure Variantions(SVs) of mother and son Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4765 Purpose: Infection is the most important cause of death in immunosuppressed cancer patients. And the urinary tract is a common source of infection in children. So we investigated the frequency of urinary tract infection (UTI) and the treatment outcome in children with cancer who were receiving antineoplastic therapy. Methods: We reviewed the medical records of children who were diagnosed as UTI during chemotherapy because of hematologic malignancies or solid tumors from January 2003 to July 2010 in Kyungpook National University Hospital. We defined as UTI when the patient showed high fever over 38.5°C and the single bacterial organism was cultured over 10,000/mL in urine sample using midstream urine collection technique. The bacterial strain, duration of fever, laboratory tests including urinalysis and gram stain, and imaging studies were demonstrated. Results: There were 63 cancer patients (male:female = 39:24) and 47 of them (74.6%, male:female = 30:17) experienced UTI during chemotherapy at least once. No one showed urinary symptom/sign like dysuria, frequency, urgency, flank pain or costo-vertebral angle tenderenss. The total episodes of UTI were 133 (male:female = 96:37) and the number of infection was mean 2.8 (1~10) per one patient. The common organism was Escherichia coli (25.6%), Enterococcus faecalis (15.0%), Klebsiella pneumoniae (10.5%), Enterococcus faecium (6.8%), Proteus mirabilis (6.8%) and Stenotrophomonas maltophilia (5.3%). Initial urinalysis was performed in 115 cases, but only 4 of them (3.5%) revealed pyuria. All gram stain results were negative. Duration of fever was mean 2.1 (1~6) days. The initial absolute neutrophil count (ANC) was average 1,930/μ L (0 ~ 12,610/μ L). The renal cortex scan using dimercaptosuccinic acid was performed for 43 cases to verify pyelonephritis. One showed decreased tracer uptake of upper pole of right kidney, and another 3 revealed diffuse decreased tracer uptake in both kidneys without specific photon defect. One episode of 3 was considered as urosepsis because the same organism (Klebsiella pneumoniae) was cultured from not only urine but also blood. The patient revealed hypotension and decreased renal function (glomerular filtration rate 41.4 mL/min). No mortality was observed. Conclusion: UTI is a very common infection in immunocompromised cancer children regardless of ANC which showed excellent prognosis with broad spectrum antibiotics. The bacterial strain was not different from that of immunocompetent children. But their symptom/sign was silent and the initial urinalysis and gram stain were not much help. So it is important to check urine culture for febrile cancer children under chemotherapy although the complication is very rare after UTI. Disclosures: No relevant conflicts of interest to declare.

Description: Biphenotypic acute leukemia (BAL) is a rare yet defined type of acute leukemia. We investigated the incidence, clinicopathologic characteristics, and clinical outcomes of BAL in Korean adults. The present study retrospectively analyzed clinicopathological and clinical data from 43 adult patients with BAL, defined using the EGIL scoring system, from 11 Korean institutes. The incidence of BAL was 2.1% among acute leukemias; 2.3% in male and 1.9% in female. Median age of 43 BAL patients, 25 males and 18 females, was 38 years (range, 16–74). The immunophenotype was myeloid/B-lymphoid (M+B) in 31 (72.1%), myeloid/T-lymphoid (M+T) in 10 (23.3%), and B/T-lymphoid (B+T) and myeloid/B/T-lymphoid (M+B+T) in one (2.3%) each. 37 patients had the results of cytogenetic analyses and Ph chromosome (n=14, 37.8%) was the single most common abnormal finding. Intensive induction chemotherapy was given in 36 of 43 patients: AML-type in 13 (36.1%), ALL-type in 8 (22.2%), and combined type in 15 (41.7%). Complete remission (CR) was induced in 29 patients (80.6%). The CR rate was significantly lower in M+T (5 of 9, 55.6%) than M+B (22 of 25, 88.0%) (P=0.039). Other unfavorable factors for CR were absence of CD19 (P=0.029) or CD20 (P=0.046), and presence of CD2 (P=0.040). The CR rates were not significantly different according to cytogenetic finding or type of induction chemotherapy. After median follow-up duration of 712 days among surviving patients, 11 patients relapsed with median relapse-free survival (RFS) of 3.08 years and 4-y RFS of 38.3% and 18 patients died with median overall survival (OS) of 2.49 years and 4-y OS of 30.1%. Multivariate analysis showed that high leukocyte counts (≥ 30,000/μl) at diagnosis (OR, 5.922; 95% CI, 1.379–25.429; P=0.017) was an independent unfavorable prognostic factor for RFS, and high leukocyte counts at diagnosis (OR, 9.348; 95% CI, 3.014–28.993; P

Description: Infection is one of the most important causes of death in cancer patients. So many physicians make every effort to control the infection, especially in neutropenic cancer patients. The aim of this study is to find out the role of HEPA filter equipped laminar air flow room reverse isolation for the management of chemotherapy induced febrile neutropenic children with cancer. We evaluated febrile neutropenic patients following chemotherapy from January 2003 to April 2006 at the Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea. They were promptly managed by antibiotics and antifungal agents and if possible, they were isolated in the aseptic room. They were allocated to three groups of standard ward care, isolation after onset of fever and isolation before onset of fever. Profiles of infection, clinical courses and survival rate were compared among three groups. One hundred and nine episodes of febrile neutropenia from thirty eight cancer patients were observed. Twenty nine were boys and nine were girls with their median age were 5.5 years. The diagnoses included acute leukemia (36.8%), malignant lymphoma (15.8%), and other solid tumor (47.4%). Fifty five episodes were included to standard ward care, forty four episodes to isolation after onset of fever and thirteen episodes to isolation before onset of fever. We found out that one in thirteen episodes of isolation before onset of fever (8%) and twenty six in ninety six episodes in other groups (27%) were microbiologically or clinically defined infections. Fifty six episodes recovered and only one died of infectious cause both isolation after and before onset of fever group, especially all recovered in isolation before onset of fever group. Forty eight episodes recovered and four died in general ward care group, but there was no statistical difference among three groups (p=0.93). Age, sexual difference, underlying disease, absolute neutrophil count (ANC), duration of ANC recovery and incidence of disseminated intravascular coagulation were no difference among three groups. In early isolated group, duration of fever and antibiotics medication were significantly shorter (p=0.002, 0.009) and CRP level was lower than other two groups (p=0.04). Reverse isolation in laminar air flow room for neutropenic cancer children before onset of fever affect the durations of fever and antibiotics treatment with beneficial effect.

Description: Abstract 2778 Poster Board II-754 Introduction: Interstitial deletions involving the long arm of chromosome 5, one of the good prognostic factors, are the most common chromosomal abnormality either as a sole or in combination with other abnormalities in myelodysplastic syndromes (MDS). However, the prognostic impact of del(5q) accompanied by additional chromosome abnormalities remains controversial. We investigated the hematologic, cytogenetic and prognostic features of del(5q) in MDS. Also, we mapped the deleted region on 5q by fluorescence in situ hybridization (FISH), whether the difference of deleted region between 5q- syndrome and MDS with del(5q) accompanied by additional abnormalities makes the clinical and prognostic differences. Methods: 137 adult patients, newly diagnosed as de novo MDS in Seoul National University Hospital from April 2000 through March 2009, were enrolled. We reclassified MDS subtypes according to WHO classification 2008. To compare the hematologic, cytogenetic and prognostic features according to presence of del(5q), we categorized the patients with del(5q) into 3 groups: patients with additional chromosomal abnormalities with del(5q) as 'MDS with del(5q)'; patients with other chromosomal abnormalities other than del(5q) as 'MDS with other chromosomal abnormalities (CA)'; and patients with isolated del(5q) as '5q- syndrome'. Also, the mapping with FISH for EGR1, CSF1R, and PDGFRβ on 5q, was performed in conjunction with G-banding to all patients and additional 16 patients with alleged del(5q) by G-banding from Korean MDS working party. Results: According to the new WHO classification of 2008, the 33 refractory anemia patients according to the previous WHO classification of 2001 were reclassified into refractory cytopenias with unilineage dysplasia (13 patients), refractory cytopenia with multilineage dysplasia (six patients) and MDS - unclassified (14 patients) (Fig 1). The median age of Korean MDS was 59 years, and the frequencies of 5q- syndrome and 5q deletion was 2.2% (3/137 patients) and 15.3%, respectively. Among 137 patients, 17 patients were grouped into 'MDS with del(5q)', and 53 patients into 'MDS with other CA'. The 'MDS with del(5q)' were significantly older and showed higher % of blasts in PB and BM than 'MDS with other CA'. And, they were categorized into higher risk group according to the International Prognostic Scoring System (IPSS) (Table 1). As a results of mapping for EGR1, PDGFRβ and CSF1R, deletion of all 3 regions was 93.3% in patients of 'MDS with del(5q)' and 66.7% in patients of '5q- syndrome', showing no difference in deleted genes between the two groups. Half (53%) of patients of 'MDS with del(5q)' accompanied complex abnormalities including chromosome 7 abnormalities. The del(5q) was detected only by FISH, showing discrepant results between G-banding and FISH analysis. Especially, marker chromosomes by G-banding in some patients were proved to be chromosome 5 with del(5q) by FISH. Conclusion: The biologic and prognostic features of MDS in Korea seem to be markedly different from those of Caucasian; younger age and low frequency of 5q- syndrome. The incidence of complex cytogenetic abnormalities including del(5q) was higher than that of Caucasian, while that of isolated del(5q) was quite low in Korea, which can explain that higher proportion of MDS with del(5q) belongs to higher risk IPSS group. And, we suggest FISH for del(5q) at initial diagnosis and during follow-up after treatment of MDS with alleged del(5q), since the presence of del(5q) in MDS is important for choosing the lenalidomide treatment. Disclosures: No relevant conflicts of interest to declare.

Description: To establish patient-derived multiple myeloma (MM) cell lines, mononuclear cells obtained from a MM patient’s bone marrow were directly injected via tail vein into a NRG/SCID mouse. Fourteen weeks after injection, tumor developed at subcutis and bone marrow (BM) in the same mouse. We separated and cultured cells from these two sites (subcutis and BM) and established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). In cytogenetic analysis, karyotype of newly established two MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In FACS analysis, the expression of CD138 and CD45 was detected in both cell lines. Response to IL-6 and soluble IL-6 receptor was different between the two cell lines. Moreover, SNU_MM1393_SC showed higher degree of resistance against proteasome inhibitor (bortezomib) compared to SNU_MM1393_BM. However, two cell lines were both sensitive to histone deacetylase inhibitor (panobinostat). When whole exome sequencing was performed using the DNA of these two cell lines, a set of somatic mutations involving Wnt signaling and NF-kB pathway were detected in both cell lines. Additional somatic mutations of JAK1, PLCG1, IRS2, and HGS which are known to interact with JAK/STAT pathway were detected in SNU_MM1393_BM. Whereas, additional somatic mutations of EGFR, HSP90AB1, CFDR, and CALML5, which are known to interact with growth factor cell signaling pathway were detected in SNU_MM1393_SC. These findings highlight the importance of interactions between tumor and tumor microenvironment as the myeloma progresses and will pave a way to more effective selection of targeted agents according to specific tumor characteristics obtained in the process of disease progression. Disclosures: No relevant conflicts of interest to declare.

Description: More than a half of anaplastic large cell lymphoma (ALCL) harbors an aberrant NPM-ALK fusion gene, which activates a number of down-stream signaling pathways such as Ras/ERK, PI3K/AKT, and JAK3/STAT3. Through this mechanism, mTOR pathway is also activated in ALK-positive ALCL (Vega F, et al. Cancer Res 2006). Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas (Jundt F, et al. Blood 2005; Wanner K, et al. Br J Haematol 2006; Haritunians T, et al. Leukemia 2007), although the clinical efficacy of everolimus monotherapy was not satisfactory, possibly due to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive tumors so far. The aim of this study was to evaluate the effect of everolimus in combination with crizotinib in ALK-positive ALCL cell lines, K-299 and SU-DHL-1. We treated K-299 and SU-DHL-1 cells with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (Figure 1). After 72 hours, the combination index (CI) values calculated by the Chou-Talalay method were less than 1 (range, 0.583-0.763 in K-299 cells and 0.271-0.616 in SU-DHL-1 cells) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. The Western blot analysis (Figure 2) demonstrated that everolimus treatment up-regulated the phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473 in K-299 cells. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited phosphorylation of mTOR Ser2448, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased phosphorylated mTOR at Ser2481, a marker for mTORC2, as well. In the cell-cycle analysis, the combination treatment induced G1 arrest. Everolimus treatment alone did not increase the fraction of cells in the sub-G1 region compared to the control (2.16% vs. 4.03% in K-299 and 1.34% vs. 1.68% in SU-DHL-1), while crizotinib monotherapy increased the sub-G1 population (11.88% vs. 4.03% in K-299 and 28.68% vs. 1.68% in SU-DHL-1). The combination of crizotinib and everolimus markedly increased the sub-G1 population in both ALK-positive ALCL cell lines (22.25% in K-299 and 46.40% in SU-DHL-1). PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib. In summary, everolimus combined with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT signaling activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis (Figure 3). Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.