ALBERT

Description: Abstract 3018 Background and Purposes Epstein-Barr virus (EBV)-associated PTLD is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Independent risk factors include use of ATG, acute GVHD, CMV antigenemia, T-depleted graft, and unrelated donor in our previous study. Quantitative real-time polymerase chain reaction (Q-PCR) was developed for early detection and intervention of EBV reactivation to prevent PTLD-related mortality. Patients and Methods Between Apr 2004 and Oct 2010, EBV viral load in plasma was monitored by Q-PCR in 222 HSCT patients (total 2945 samples) in NTUH. EBV reactivation was defined as 〉 500 copies/mL in two consecutive assays or 〉 10-fold elevation than baseline level. Results EBV reactivation occurred in 50 (22%) patients. The cumulated incidence of EBV reactivation was 28% at 1-year and 32% at 2-year. Median time to EBV reactivation was 40 days (ranges, 26–406) after SCT and median peak EBV-viral load, 10888 copies/ml (ranges, 948–3×107). The risk of EBV reactivation was significantly higher in patients receiving ATG (52% vs. 13%, p〈 0.001), use of TBI (58% vs. 26%, p

Description: Abstract 5129 Introduction Preeclampsia is a pregnancy-induced disorder. If a pregnant woman suffered from preeclampsia, her worse developed-placenta might result in growth retardation of the fetus, and in the most severe cases, it would cause fetal and maternal death. Several previous studies showed that circulating thromboxane A2 in preeclampsic women was higher than that of normal pregnant women, and higher thromboxane A2 level might induce thrombosis and hypertension in these patients. Thus, thromboxane A2 was thought to be an important metabolite relevant to the development of preeclampsia. Materials and Methods In order to clearly clarify the roles of thromboxane A2 on preeclampsia, wild type and thromboxane A2 synthase knockout pregnant mice were fed with drinking water containing high concentration of sodium chloride. The effect of thromboxane A2 on preeclampsia was observed by measuring blood pressure, urinary protein, systemic edema in pregnant mice, and placenta weight, fetal weight, fetal size and fetal sections were also measured and stained. Results In the groups of pregnant mice fed with high salt, maternal body weight, fetal size and fetal weight were severely decreased in wild type mice as compared to thromboxane A2 synthase knockout mice. Furthermore, abnormal cell degeneration was observed in fetus of wild type pregnant mice, but less in thromboxane A2 synthase knockout fetus. Conclusions These results demonstrate that knockout pregnant mice were more resistant to high salt treatment, implying the roles of thromboxane A2 on the development of maternal and fetal defects during high salt treatment. Disclosures No relevant conflicts of interest to declare.

Description: Neutrophils are the first cells to arrive at the site of infection where they play a critical role in host defense against invading microbes. To perform this process, neutrophils produce and secrete a lot of molecules to destroy the infectious agent. Among proteolytic enzymes identified in the neutrophils, matrix metalloproteinases (MMPs) have attracted considerable attention for their proposed role in the destruction of extracellular matrix under physiological and pathological conditions. The first MMP identified in neutrophils is MMP-8 or neutrophil collagenase that is stored in the specific granules and is capable of cleaving type I collagen. MMP-9, neutrophil gelatinase, are stored in the gelatinase granules and is capable of cleaving type VI collagen. Chronic myeloid leukemia (CML) is a malignant clonal disorder that originates from a transformed stem cell and involves myeloid lineage. The affected cells have both proliferative and functional impairment. Leukemoid reaction (LR) often encountered in association with severe infections or malignancy, shows extreme elevations of the leukocyte count with left shift including myeocytes, promyelocytes and blasts that would be confused with CML. The laboratory differential diagnosis of CML and LR are based on the leukocyte alkaline phosphatase (LAP) score, philadelphia chromosome analysis and bone marrow analysis. Our previous study revealed that the level of marrow MMP-9 may be a useful surrogate marker for monitoring disease status in AML and propose it as a potential prognostic factor. In this study, CML patients at diagnosis, various patients with leukemoid reaction and several normal individuals were assessed, to evaluate the potentiality of MMP-9 or/and MMP-8 to be differential diagnostic markers between CML and LR. We determined the MMP-8 and MMP-9 concentrations in peripheral blood by ELISA method, and the MMP-8 and MMP-9 contents in neutrophils by immunocytochemical staining and flow cytometry. We found that the plasma MMP-8 levels were less than 10ng/ml in all normal individuals studied, those were 10 – 100ng/ml in CML patients and those were more than 100ng/ml in LR patients. The same trend was shown in MMP-9 levels, which were less than 50ng/ml, 50 – 450ng/ml and more than 450ng/ml, respectively. Immunocytochemical staining revealed MMP-8 and MMP-9 contents in cytoplasm of neutrophils were abundant in patients with LR, compared with those in patients with CML and normal individuals; further flow cytometric analysis also showed the same trend. These results suggest that the MMP-8 and MMP-9 concentrations and individual neutrophil MMP-8 and MMP-9 contents might contribute to discriminate between CML and LR, and to be useful surrogate markers for differential diagnosis.

Description: Key Points We identified 3 novel loss-of-function variants in NUDT15 linked to thiopurine intolerance. Our findings extended the importance of NUDT15 variation in thiopurine pharmacogenetics in diverse populations.

Description: In adaptation of risk-directed combined chemotherapies, the initial remission rate in treatment of childhood acute lymphoblastic leukemia (ALL) has exceeded 95%. Hematological relapse during maintenance therapy, in which methotrexate (MTX) and thiopurine are applied, is the major cause of treatment failure. A retrospective study was performed to evaluate the role of pharmacogenomic effects in the treatment of children with ALL in the southern Chinese population. A total of 105 Taiwanese children with ALL, who received combined chemotherapy of different intensities based on risk-directed Taiwan Pediatric Oncology Group (TPOG)-ALL-93 protocols between Oct. 1993 to Dec. 2001, were recorded in long-term follow-up (6.5 to 13.7 years) for events (hematological relapse or death) occurrence (Figure 1). Seventeen genetic polymorphisms in 13 pharmacogenomic targets that implicated in MTX/thiopurine metabolism were analyzed by PCR-based restriction length polymorphism (RFLP) or sequence-specific oligonucleotide (SSO) probe hybridization. Pharmacogenomic polymorphisms were correlated with long-term event-free survival (EFS) of patients, with confounding effects adjusted by multivariate regression. Homozygosity of the 2677–3435 G-C allele in the multi-drug resistance gene (MDR-1, ABCB1) was highly associated with a significant reduction in long-term EFS in those patients treated with the standard risk protocol (TPOG-ALL-93-SR) (Figure 2). In the 36 patients receiving TPOG-ALL-93-SR treatment protocol, 6 out of 12 (50%) subjects carried homozygotic MDR1 2677–3435 G-C/G-C genotype suffered hematological relapse in 2 years, compared to 21 of 24 (88%) the non-homozygotic subjects remained event-free after 5 years (hazard ratio: 6.8, p=0.01). Among patients treated with the a high risk protocol (TPOG-ALL-93-HR) due to the presence of myeloid markers on the leukemic cells or manifested central nervous system leukemia, the thymidylate synthase (TYMS) enhancer 28-bp repeats 3R3R, and the glutathione-S-transferase M1 (GSTM1) null genotypes were associated with inferior clinical outcomes (p=0.029 and 0.058, respectively). Moreover, for patients with T-cell ALL that received the very high risk protocol (TPOG-ALL-97-VHR), the methionine synthase reductase (MTRR) 66AA genotype correlated with a superior prognosis compared to the AG or GG genotypes. These findings indicated independent pharmacogenomic determinants could be identified in subsets of Taiwanese children with ALL and correlated to the treatment outcome. In conclusion, we propose the pharmacogenomic determinants disclosed in the context of TPOG-ALL-93 protocols could be used to refine protocols for the treatment of pediatric ALL patients. Fig. 1 Kaplan-Meier plot depicting event-free survival of the ALL patients. The estimate of five-year event-free-survival (and the standard error) is illustrated for 105 patients received TPOG-ALL-93 protocols. Fig. 1. Kaplan-Meier plot depicting event-free survival of the ALL patients. The estimate of five-year event-free-survival (and the standard error) is illustrated for 105 patients received TPOG-ALL-93 protocols. Fig. 2 MDR1 2677–3435 G-C/G-C genotype identified a subset with poor prognosis in the 36 ALL patients received TPOG-ALL-93-SR treatment protocol. Fig. 2. MDR1 2677–3435 G-C/G-C genotype identified a subset with poor prognosis in the 36 ALL patients received TPOG-ALL-93-SR treatment protocol.

Description: MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are regulators of gene expression. A role for microRNAs in leukemia, such as chronic lymphoblastic leukemia, acute myelogeneous leukemia has recently been recognized. However, little is known about the role of microRNAs in childhood acute lymphoblastic leukemia (ALL). To determine whether miRNAs are associated with the clinical features of childhood ALL and its association with cytogenetic abnormalities, we analyzed 60 untreated childhood ALL cases for their miRNA expression using a microarray platform. Leukemic samples were collected from the ALL children between 1–18 years of age. Of the 365 miRNA analyzed with a training group of 40 patients, a miRNA signature was derived that was associated with event-free survival. The signature was tested in a validation group of 20 patients. For the latter, a miRNA compound covariate predictor (i.e., a miRNA risk score) was computed on the basis of weighted levels of the miRNAs forming the outcome signature. The signature identified from the training group contained 5 miRNA highly associated with event-free survival (P

Description: Introduction The nationwide TPOG-ALL-2002 protocol for treating children with acute lymphoblastic leukemia (ALL) was activated in January 2002 in Taiwan. To eliminate cranial radiation (CrRT)-related sequelae and minimize the adverse prognostic impact of traumatic lumbar puncture with blasts (TLP), we conducted a prospective study beginning in 1999 to modify CNS-directed therapy with delayed first triple intrathecal therapy (TIT) and omission of CrRT for all risk groups of newly diagnosed ALL. This approach improved CNS control at the single-institution study (J Clin Oncol, 2014) and has been used in whole TPOG since January 2009. Patients with non-CNS-1 status (i.e., CNS-2, CNS-3 and TLP) have higher risk of CNS relapse and need intensive CNS therapy. On behalf of TPOG members, we reported the outcomes of these non-CNS-1 patients in the two eras (2002-2008 and 2009-2012) with or without the modified CNS therapy. Methods From 2002 to 2012, all newly diagnosed ALL children in Taiwan were enrolled in TPOG-ALL-2002 protocol (Leukemia, 2010). B-precursor ALLs were stratified into standard risk (SR), high risk (HR) and very high risk (VHR) groups, according to age, WBC count, cytogenetics and molecular analysis at diagnosis. T-cell ALL was designated as VHR. Since 2009, patients had been treated with TIT alone without CrRT. The first TIT was performed until the disappearance of blasts from peripheral blood (PB), no later than 10 days. SR patient with detectable PB blasts on day 8 was upgraded to HR group. SR with CNS-1 received 14 doses of TIT, and those with CNS-2, or TLP got 2 additional TITs during induction and 3 additional TITs during maintenance therapy. Totally, SR with CNS-2 or TLP received 19 doses of TIT. And SR patients with CNS-3 or cranial nerve palsy at diagnosis were classified as HR. For HR with CNS-1, 17 doses of TIT were given. If HR groups with CNS-2, CNS-3, or TLP, they will receive total 25 doses of TIT including 2 additional ones during induction and 6 additional ones during maintenance therapy. Before 2009, only SR and HR with refractory CNS leukemia (failure to clear CSF blasts after 3 consecutive TITs) and those with CNS relapse will receive CNS radiation. For VHR group before 2009, CrRT of 18 Gy was given to all patients at the end of remission induction. Instead, TIT was given for age 〈 2 years. After 2009, CrRT was totally omitted. VHR with CNS-1 received 19 doses of TIT. For VHR patients with CNS-2, CNS-3 or TLP at diagnosis, TIT was administered once/week until achieving CSF remission and then once 4 weeks during maintenance therapy. Results Since January 2009, all patients received TIT alone, irrespective of risk groups. The two cohorts, totally 1,366 patients, 909 treated with CrRT (2002-2008) and 457 without CrRT (2009-2012) were balanced in presenting features and distribution of CNS status. There were no significant differences between two eras in the event-free survival (EFS), overall survival (OS), incidences of isolated CNS relapse and any CNS relapse (Pediatr Blood Cancer, accepted June 2016). The number of patients with CNS-2, CNS-3 and TLP were 35, 26 and 19 before 2009; and 18, 10 and 9 after 2009, respectively. Patients with younger age (〈 1 year), higher WBC counts (〉 100 X 109/L), HR/VHR or MLL rearrangement had significantly more non-CNS-1 status in the era of 2002-2008. And the significant factors prone to be non-CNS-1 were male, T-lineage and HR/VHR with the first TIT delaying. There were no significant differences between the two eras in the 5-year EFS and OS. Though the incidence of 5-year any CNS relapse was not significantly different, fewer relapses were observed in the era of 2002-2008 (Table 1). Before 2009, eleven CNS relapses experienced between 1 month and 3.1 years (median, 10 months) after remission. Two relapses after 2009 occurred on 1 month and 7 months, respectively. Conclusions Delayed first TIT until the clearance of circulating blasts and total omission of CrRT did not compromise the survivals and CNS control of childhood ALL patients with CNS-2, CNS-3 and traumatic lumbar puncture with blasts. Moreover, this approach prevents the adverse sequelae caused by CrRT. Furthermore, a skillful TIT is mandatory to improve results. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2505 Background: Genetic alterations affecting the B cell transcription factor PAX5 were identified in over 30% of leukemic samples obtained from both pediatric and adult pro-B ALL patients in previous studies using high-resolution single-nucleotide polymorphism (SNP) microarrays. The clinical significance of PAX5 hemizygous deletion has not been fully characterized yet. In order to understand the role of PAX5 alteration in the context of pro-B ALL, we investigated the genetic changes as well as the relevant PAX5 transcript/protein expression in the leukemic cells. Patients and methods: Twenty-four children with pro-B ALL enrolled in Taiwan Pediatric Oncology Group (TPOG) were studied. RNA samples were prepared from the diagnostic bone marrow and PAX5 mRNA expression was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). The PCR products were subjected to cloning and nucleotide sequence analysis to detect mutations/micro-deletion in the PAX5 coding transcripts. PAX5 protein expression in leukemic cells was analyzed by Western blotting in 6 available samples. Mature B cells purified from PBMCs of healthy subjects were served as control in RT-PCR and Western blotting assays. Variation of copy number in PAX5 gene was analyzed in the above 6 patients. To assess the loss of heterozygosity (LOH) at/near PAX5 locus (PAX5/LOH) in the leukemic cells, length polymorphisms in 5 micro-satellite (STR) markers spanning chromosome 9p were compared between the leukemic cells and respective PBMCs obtained at the remission stage. Results: PAX5 transcripts were expressed at a reduced level (less than half of that in the normal mature B cells) in 13 of the 24 leukemic samples. While performing RT-PCR spanning exons 6 to 10 of PAX5, PCR products of different size (corresponding to full length, -E8, -E7/8, -E8/9 etc.) were frequently identified in the leukemic samples, compared to primarily full length- and little -E8-transcripts shown in the normal B cells. We did not identify any mutations or micro-deletion in the amplified PAX5 transcripts. In six leukemic samples available for both PAX5 protein Western blotting and DNA PAX5/LOH analysis, three showed a massive reduction in PAX5 protein while another 3 express PAX5 protein comparable to that of the normal mature B cells. The reduced PAX5 protein production correlated to the reduced level of full-length PAX5 transcripts. Three leukemic samples showed LOH at/near PAX5 locus. However, PAX5/LOH did not correlate with the reduced PAX5 mRNA and protein expression in the leukemic cells: 2 leukemic samples with PAX5/LOH showed PAX5 expression comparable to that of the normal mature B cells. Development of non-B-lineage differentiation was demonstrated in a mouse model with a conditionally knocked-out PAX5 in the committed B-cells. Although statistically non-significant, presence of non-B-lineage differentiation markers was more frequently present in the leukemic cells reduced PAX5 expression. Conclusion: LOH at or near PAX5 locus is common, while point mutation or micro-deletion in PAX5 is rarely found in childhood pro-B ALL. Reduced PAX5 expression (in mRNA and protein) is common in leukemic cells. However, it does not correlate to the PAX5 hemizygous deletion status in the corresponding cells. Mechanisms other than haplo-insufficiency might determine PAX5 expression in the pro-B ALL cells. In leukemic samples, different variant forms at the C-terminal part of PAX5 transcript were frequently identified. The significance of the reduced PAX5 expression and the presence of the variant PAX5 transcripts, in leukemogenesis and the clinical settings in pro-B ALL remained to be studied. Disclosures: No relevant conflicts of interest to declare.